Human CDR-grafted antibody and antibody fragment thereof

ABSTRACT

A human CDR-grafted antibody or the antibody fragment thereof which specifically reacts with the extracellular region of human CC chemokine receptor 4 (CCR4) but does not react with a human blood platelet; a human CDR-grafted antibody or the antibody fragment thereof which specifically reacts with the extracellular region of CCR4 and has a cytotoxic activity against a CCR4-expressing cell; and a medicament, a therapeutic agent or a diagnostic agent comprising at least one of the antibodies and the antibody fragments thereof as an active ingredient.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 13/428,739 filedMar. 23, 2012, which is a divisional of U.S. application Ser. No.12/903,780 filed Oct. 13, 2010(U.S. Pat. No. 8,143,058), which is adivisional of U.S. application Ser. No. 12/395,214filed Feb. 27, 2009(U.S. Pat. No. 7,842,797), which is a divisional of application Ser. No.10/231,452 filed Aug. 30, 2002 (U.S. Pat. No. 7,504,104). The entiredisclosure of prior applications is considered part of the disclosure ofthe accompanying divisional application and are hereby incorporated byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a human CDR-grafted antibody whichspecifically reacts with the extracellular region of human CC chemokinereceptor 4 (hereinafter referred to as “CCR4”) but does not react with ablood platelet and the antibody fragment thereof. Furthermore, thepresent invention relates to a human CDR-grafted antibody whichspecifically reacts with the extracellular region of human CCR4 but doesnot have an inhibiting activity of a CCR4 ligand such as TARC or MDCbinding to CCR4 and the antibody fragment thereof. Moreover, the presentinvention relates to a human CDR-grafted antibody which specificallyreacts with the extracellular region of CCR4, has a cytotoxic activityand an inhibiting activity of cytokine production by Th2 cells, andcomprises a specific complementarity determining region (hereinafterreferred to as “CDR”), and the antibody fragment thereof. Also, thepresent invention relates to a DNA encoding the antibody or the antibodyfragment thereof. Furthermore, the present invention relates to a vectorcomprising the DNA, and a transformant transformed with the vector.Moreover, the present invention relates to a method for producing theantibody or the antibody fragment thereof using the transformant, and amedicament such as a therapeutic agent, a diagnostic agent and the like,for Th2-mediated immune diseases such as allergic diseases and the like,which comprises using the antibody or the antibody fragment thereof.Additionally, the present invention relates to a medicament such as atherapeutic agent, a diagnostic agent and the like, for cancers such asblood cancers, e.g., leukemia and lymphomatosis, which comprises usingthe antibody or the antibody fragment thereof.

2. Brief Description of the Background Art

Various factors such as eosinophils, mast cells, IgE and the like, playa role in allergic diseases such as bronchial asthma. Eosinophilsinfiltrate into an inflammatory site and release cytotoxic basicproteins such as MBP (major basic protein) or the like by degranulationand the surrounding tissues are damaged by such cytotoxic basicproteins. Mast cells are bound to an antigen immune complex with IgEwhich is produced by B cells and release histamine so that an immediateallergic reaction is induced. The allergic reaction is controlled bybiologically functional molecules such as cytokines, chemokines, and thelike, which take part in signal transduction between cells. IL-5 inducesdifferentiation, survival and degranulation of eosinophils. IL-4 inducesB cell activation and production of IgE. IgE produced forms an immunecomplex with the antigen and causes degranulation of mast cells. It hasbeen found that IL-4, IL-13 and the like are also produced by mast cellsand contribute to the production of IgE by B cells (Am. J. Respir. Crit.Care Med., 152, 2059 (1995), Immunol. Today, 15, 19 (1994)). Thus, anelaborate cytokine-chemokine network is present among inflammatory cellsand keeps complicated balances.

The cytokines and chemokines are produced by helper T cells whichexpress CD4 on the cell surface (hereinafter referred to as “CD4+ Thcells”). Actually, it has been found that infiltration of helper T cellsis found in the airway inflammation site of bronchial asthma patients,wherein a considerably large number of the T cells are activated andthat the severity and airway hypersensitivity of asthma patientcorrelates with the number of activated T cells, as well as theactivated T cells are also increased in the peripheral blood (Am. Rev.Respir. Dis., 145, S22 (1992)).

The helper T cells are classified into Th1 cells and Th2 cells,depending on the kind of cytokine to be produced thereby (Annu. Rev.Immunol., 7, 145 (1989)). Th2 cells produce IL-4, IL-5, IL-13 and thelike. The cytokines produced by Th2 cells are Th2 cytokines.

It has been found that an antigen-specific T cell clone isolated from anatopic disease patient releases Th2 cytokines when stimulated in vitro(Proc. Natl. Acad. Sci., U.S.A., 88, 4538 (1991)), and Th2 cells arepresent in bronchoalveolar lavage fluid (hereinafter referred to as“BAL”) and airway mucosa of asthma patients (N. Engl. J. Med., 326, 298(1992), Eur J. Immunol., 23, 1445 (1993)). Expression levels of IL-4 andIL-5 mRNAs of Th2 cytokines are increased when mRNA expressions ofvarious cytokines in cells in BAL are examined using an allergicinflammation animal model (Clin. Immunol. Immunopathol., 75, 75 (1995)).Also, when Th2 cells are intravenously or intranasaly administered tomice, asthmatic inflammation is induced in the lungs in antigen specificmanner (J Exp Med., 186, 1737 (1997), J. Immunol., 160, 1378 (1998)) andeosinophilia is observed (J. Immunol., 161, 3128 (1998)). Expression ofIL-5 is observed in the airway mucous of asthma patients and the skinlesions of atopic dermatitis patients (J. Clin. Invest., 87, 1541(1991), J Exp. Med., 173, 775 (1991)), and the expression level of IL-5in the mucous of chronic allergic rhinitis correlates with theexpression level of IL-13, and the amounts of serum total IgE andantigen-specific IgE (Therapeutics, 32, 19 (1998)).

Chemokine is a general term for basic heparin-binding proteins whichinduce chemotoxis and activation of leukocyte, and classified intosubfamilies of CXC, CC, C and CX₃C depending on the position of thecysteine residues in the primary structure. 16 of chemokine receptorshave been identified so far (Curr. Opi. Immunol., 11, 626 (1999)), andit has been shown that expression of each chemokine receptor isdifferent among the leukocytes such as Th1 cell, Th2 cell or the like(Cell Engineering, 17, 1022 (1998)).

Human CCR4 is a G protein coupled seven transmembrane receptor cloned asK5-5 from a human immature basophilic cell line KU-812. Thetransmembrane regions of CCR4 are considered to be positions 40 to 67,positions 78 to 97, positions 113 to 133, positions 151 to 175,positions 207 to 226, positions 243 to 270 and positions 285 to 308 inthe amino acid sequence, the extracellular regions are considered to bepositions 1 to 39, positions 98 to 112, positions 176 to 206 andpositions 271 to 284 in the amino acid sequence, and the intracellularregions are positions 68 to 77, positions 134 to 150, positions 227 to242 and positions 309 to 360 in the amino acid sequence (J. Biol. Chem.,270, 19495 (1995)). At first, it was reported that the ligand of CCR4 isMIP-1α (macrophage inflammatory protein-1α), RANTES (regulated onactivation normal T-cell expressed and secreted) or MCP-1 (monocytechemotactic protein) (Biochem. Biophys. Res. Commun., 218, 337 (1996),WO 96/23068). However, it has been found that TARC (thymus andactivation-regulated chemokine) produced from stimulated humanperipheral blood mononuclear cells (hereinafter referred to as “PBMC”)and thymus cells (J. Biol. Chem., 271, 21514 (1996)) specifically bindsto CCR4 (J. Biol. Chem., 272, 15036 (1997)). It has been also reportedthat MDC (macrophage-derived chemokine) isolated from macrophage (J.Exp. Med., 185, 1595 (1997)), also known as STCP-1 (stimulated T cellchemotactic protein-1) (J. Biol. Chem., 272, 25229 (1997)), binds toCCR4 more strongly than TARC (J Biol. Chem., 273, 1764 (1998)).

It has been shown that CCR4 is expressed on CD4+ Th cells which producecytokine and/or chemokine (J. Biol. Chem., 272, 15036 (1997)), and ithas been reported that CCR4 is expressed selectively on Th2 cells amongCD4+ Th cells (J. Exp. Med., 187, 129 (1998), J. Immunol., 161, 5111(1998)). In addition, CCR4+ cells have been found in effector/memory Tcells (CD4+/CD45RO+), and when CCR4+ cells were stimulated, IL-4 andIL-5 were produced but IFN-γ was not produced (Int. Immunol., 11, 81(1999)). Also, it has been reported that CCR4+ cells belong to a CLA(cutaneous lymphocyte antigen)-positive and α4β8 integrin-negative groupamong memory T cells, and CCR4 is expressed on memory T cells relatednot to gut immunity but to systemic immunity of the skin and the like(Nature, 400, 776 (1999)). These results strongly suggest that wheninflammation is induced, the memory T cells are activated to expressCCR4 and are migrated into the inflammatory site by MDC and TARC ofligands of CCR4, and accelerate activation of other inflammatory cells.

It has been recently found that CCR4 is also expressed in natural killercells (Journal of Immunology, 164, 4048-4054 (200), Blood, 97, 367-375(2001)) and platelets (Thrombosis Research, 101, 279-289 (2001), Blood,96, 4046-4054 (2000), Blood, 97, 937-945 (2001)) in human.

It is known that an antagonist of TARC or MDC as a ligand of CCR4,namely a CCR4 antagonist, inhibits platelet aggregation (WO 99/15666).It is known that such an agent modulating the function of CCR4 alsoaffects on platelet functions.

Expression of CCR4 is found in platelets. For example, Adrian et al.(Blood, 97, 937-945 (2001)) and Abi-Younes et al. (Thrombosis Research,101, 279-289 (2001), WO 00/42074, WO 00/41724) have detected expressionof CCR4 in human platelets using anti-CCR4 antibodies. An antibodyhaving reactivity with CCR4 but not capable of binding to humanplatelets has not been known to date.

Also, it is known that when an autoantibody capable of binding toplatelets is produced, autoimmune thrombopenia is induced (Blood, 70,428-431 (1987), Transfusion Science, 19, 245-251 (1998)). Agents havinginfluences on thrombocytopenia and platelet functions are not generallydesirable as medicaments because they often cause severe side effectssuch as bleeding and thrombus formation. Particularly, since antibodieshave long blood half-life, an antibody which affects on plateletfunctions is difficult to be developed as a medicament. For example,development of anti-CD40 ligand antibodies which had been developed asagents for treating autoimmune diseases has been suspended because ofthe generation of side effects which are considered to be due torecognition of antigen expressed on activated platelets (NatureMedicine, 6, 114 (2000), BioCentury, A8 of 18 (2002 Jun. 20)).

It is known that proteins are subjected to various modificationreactions after translation. One of the modification reactions known isa sulfated reaction of tyrosine residues. It has been reported that manyproteins are sulfated at tyrosine residues (Chemistry and Biology, 7,R57-R61 (2000)). The tyrosine residue which is sulfated hascharacteristic that many acidic amino acid residues are present in itsvicinity, and a protein having a possibility of being sulfated and itsregion have been suggested (Cell, 96, 667-676 (1999)). Regarding CCR4, 4tyrosine residues are present close to the N-terminal, but there are noreports showing that the tyrosine residues are sulfated.

As the current method for treating Th2-mediated immune diseases, thefollowings have been developed: (1) antagonists for cytokines andchemokines such as a humanized anti-IL-5 antibody (SB-240563: SmithKline Beecham, Sch-55700 (CDP-835): Shehling Plough/Celltech), ahumanized IL-4 antibody (U.S. Pat. No. 5,914,110), a soluble chemokinereceptor (J. Immunol., 160, 624 (1998)), etc.; (2) cytokine/chemokineproduction inhibitors such as an IL-5 production inhibitor (JapanesePublished Unexamined Patent Application No. 53355/96), a retinoidantagonist (WO 99/24024), splatast tosilate (IPD-1151T, manufactured byTaiho Pharmaceutical), etc.; (3) agents acting on eosinophil, mast celland the like as final inflammatory cells, such as a humanized IL-5receptor antibody (WO 97/10354), a CC chemokine receptor 3 (CCR3)antagonist (Japanese Published Unexamined Patent Application No.147872/99), etc.; (4) inflammatory molecule inhibitors such as ahumanized anti-IgE antibody (Am. J. Respir. Crit. Care Med., 157, 1429(1998)), etc.; and the like. But they inhibit only a part of theelaborate network among cytokine, chemokine and inflammatory cells.Th2-mediated immune diseases should not be cured by these agents.Anti-CD4 antibodies have an activity to control T cells, and haveeffects on steroid-dependent severe asthma. However, since the CD4molecule is broadly expressed in various immune cells, they lack inspecificity and have a drawback of accompanying strong immunosuppressiveeffect (Int. Arch. Aller. Immunol., 118, 133 (1999)).

Thus, in order to inhibit all of them, it is required to controlspecifically upstream of the problematic allergic reaction, namely Th2cells.

The currently used common method for treating patients of severeTh2-mediated immune diseases is steroid administration, but side effectsby steroids cannot be avoided. Also, there are drawbacks that theconditions of each patient return to the former state when the steroidadministration is suspended, and that drug resistance is acquired whenthe steroid is administered for a long time.

To date, no human CDR-grafted antibody and the antibody fragment thereofwhich can detect CCR4-expressing cells and also has cytotoxicity againstCCR4-expressing cells has been established. In addition, no therapeuticagent which can inhibit production of Th2 cytokine has been known sofar.

Although it has been reported that CCR4 is also expressed on the cancercells of leukemia patients (Blood, 96, 685 (2000)), no therapeutic agentwhich depletes leukemia cells has been known.

It is known in general that when an antibody derived from a non-humananimal, e.g., a mouse antibody, is administered to human, it isrecognized as an foreign substance and induces a human antibody againstthe mouse antibody (human anti-mouse antibody: hereinafter referred toas “HAMA”) in the human body. It is known that the HAMA reacts with theadministered mouse antibody to cause side effects (J. Clin. Oncol., 2,881 (1984), Blood, 65, 1349 (1985), J. Natl. Cancer Inst., 80, 932(1988), Proc. Natl. Acad. Sci. USA., 82, 1242 (1985)), to acceleratedisappearance of the administered mouse antibody from the body (J. Nucl.Med, 26, 1011 (1985), Blood, 65, 1349 (1985), J. Natl. Cancer Inst., 80,937 (1988)), and to reduce therapeutic effects of the mouse antibody (J.Immunol., 135, 1530 (1985), Cancer Res., 46, 6489 (1986)).

In order to solve these problems, attempts have been made to convert anantibody derived from a non-human animal into a human CDR-graftedantibody using genetic engineering technique.

The human CDR-grafted antibody is an antibody in which the amino acidsequence of CDR in the variable region (hereinafter referred to as “Vregion”) derived from a non-human animal antibody is grafted into anappropriate position of a human antibody (Nature, 321, 522 (1986)). Incomparison with antibodies derived from non-human animals such as mouseantibodies and the like, these human CDR-grafted antibodies have variousadvantages for clinical applications to human. For example, it has beenreported that its immunogenecity was reduced and its blood half-lifebecame long compared with a mouse antibody using a monkey (Cancer Res.,56, 1118 (1996), Immunol, 85, 668 (1995)). Thus, it is expected that thehuman CDR-grafted antibodies have less side effects in human and theirtherapeutic effects continue for a longer time than antibodies derivedfrom non-human animals.

Furthermore, since the human CDR-grafted antibody is prepared by usinggenetic engineering technique, molecules in various forms can beprepared. For example, when γ1 subclass is used as a heavy chain(hereinafter referred to as “H chain”) constant region (hereinafterreferred to as “C region”) (H chain C region is referred to as “CH”) ofa human antibody, a humanized antibody having a high effector functionsuch as antibody-dependent cell-mediated cytotoxic (hereinafter referredto as “ADCC”) activity can be prepared (Cancer Res., 56, 1118 (1996))and a prolonged blood half-life compared with a mouse antibody isexpected (Immunol., 85, 668 (1995)). Also, in treatment particularly forreducing the number of CCR4-expressing cells, higher cytotoxicactivities such as complement-dependent cytotoxic activity (hereinafterreferred to as “CDC activity”) and ADCC activity via the Fc region (theregion in and after the hinge region of an antibody heavy chain) of anantibody are important for the therapeutic effects. Therefore, theseresults clearly show that human CDR-grafted antibodies are preferred toantibodies derived from non-human animals such as mouse antibodies.

Furthermore, according to the recent advances in protein engineering andgenetic engineering, antibody fragments having a smaller molecularweight such as Fab, Fab′, F(ab′)₂, a single chain antibody (hereinafterreferred to as “scFv”) (Science, 242, 423 (1988)), a dimerized V regionfragment (hereinafter referred to as “Diabody”) (Nature Biotechnol., 15,629 (1997)), a disulfide stabilized V region fragment (hereinafterreferred to as “dsFv”) (Molecular Immunol, 32, 249 (1995)), a peptidecontaining CDR (J. Biol. Chem., 271, 2966 (1996)) and the like, can beprepared as human CDR-grafted antibodies. The antibody fragments areexcellent in transitional activity into target tissues compared tocomplete antibody molecules (Cancer Res., 52, 3402 (1992)).

It is considered that these fragments derived from human CDR-graftedantibodies and antibody fragments thereof are more desirable than thosederived from antibodies derived from non-human animals such as mouseantibodies, when used in clinical applications to human.

As described above, diagnostic and therapeutic effects can be expectedfrom human CDR-grafted antibodies and antibody fragments thereof whenused alone, but attempts have been made to further improve the effectsby using other molecules in combination. For example, cytokine can beused as one of such molecules. Cytokine is a general term for varioussoluble factors which control intercellular mutual functions in immunereactions. CDC activity and ADCC activity, for example, are known as thecytotoxic activities of antibodies, and ADCC activity is controlled byeffector cells having Fc receptors on the cell surface such asmonocytes, macrophages, NK cells and the like (J. Immunol., 138, 1992(1987)). Since various cytokines activate these effector cells, they canbe administered in combination with an antibody in order to improve ADCCactivity of the antibody and the like.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a novel humanCDR-grafted antibody which specifically reacts with an extracellularregion of CCR4 or the antibody fragment thereof.

The present invention relates to the following (1) to (59).

(1) A human CDR-grafted antibody which specifically reacts with anextracellular region of human CC chemokine receptor 4 (CCR4) but doesnot react with a human platelet, or the antibody fragment thereof.

(2) A human CDR-grafted antibody which specifically reacts with anextracellular region of human CC chemokine receptor 4 (CCR4) and has acytotoxic activity against a CCR4-expressing cell, or the antibodyfragment thereof.

(3) The human CDR-grafted antibody or the antibody fragment thereofaccording to (1), which has a cytotoxic activity against aCCR4-expressing cell.

(4) The human CDR-grafted antibody or the antibody fragment thereofaccording to any one of (1) to (3), wherein the extracellular region isan extracellular region selected from the group consisting of positions1 to 39, 98 to 112, 176 to 206 and 271 to 284 in the amino acid sequencerepresented by SEQ ID NO:48.

(5) The human CDR-grafted antibody or the antibody fragment thereofaccording to any one of (1) to (4), wherein the extracellular region isan epitope present in positions 2 to 29 in the amino acid sequencerepresented by SEQ ID NO:48.

(6) The human CDR-grafted antibody or the antibody fragment thereofaccording to any one of (1) to (5), wherein the extracellular region isan epitope present in positions 13 to 29 in the amino acid sequencerepresented by SEQ ID NO:48.

(7) The human CDR-grafted antibody or the antibody fragment thereofaccording to any one of (1) to (6), wherein the extracellular region isan epitope present in positions 13 to 25 in the amino acid sequencerepresented by SEQ ID NO:48.

(8) The human CDR-grafted antibody or the antibody fragment thereofaccording to any one of (1) to (7), which specifically reacts with aCCR4-expressing cell.

(9) The human CDR-grafted antibody or the antibody fragment thereofaccording to any one of (1) to (8), which has a higher cytotoxicactivity against a CCR4-expressing cell than a monoclonal antibodyproduced by a non-human animal hybridoma.

(10) The human CDR-grafted antibody or the antibody fragment thereofaccording to any one of (2) to (9), wherein the cytotoxic activity is anantibody-dependent cell-mediated cytotoxic (ADCC) activity.

(11) The human CDR-grafted antibody or the antibody fragment thereofaccording to (10), wherein the ADCC activity is an activity of inducingapoptosis of a CCR4-expressing cell.

(12) The human CDR-grafted antibody or the antibody fragment thereofaccording to any one of (1) to (11), which has an activity of depletinga CCR4-expressing cell.

(13) The human CDR-grafted antibody or the antibody fragment thereofaccording to any one of (8) to (12), wherein the CCR4-expressing cell isa Th2 cell.

(14) The human CDR-grafted antibody or the antibody fragment thereofaccording to any one of (1) to (13), which has an activity of inhibitingcytokine-production of a Th2 cell.

(15) The human CDR-grafted antibody or the antibody fragment thereofaccording to (14), wherein the cytokine is IL-4, IL-5 or IL-13.

(16) The human CDR-grafted antibody or the antibody fragment thereofaccording to any one of (1) to (15), which belongs to a human IgGantibody.

(17) The human CDR-grafted antibody or the antibody fragment thereofaccording to any one of (1) to (16), which comprises complementaritydetermining regions (CDRs) of a heavy chain (H chain) variable region (Vregion) and a light chain (L chain) V region of a monoclonal antibodyagainst CCR4.

(18) The human CDR-grafted antibody or the antibody fragment thereofaccording to any one of (1) to (17), which comprises complementaritydetermining regions (CDR) of a heavy chain (H chain) variable region (Vregion) and a light chain (L chain) V region of a monoclonal antibodyagainst CCR4, and framework regions (FRs) H chain V region and L chain Vregion of a human antibody.

(19) The human CDR-grafted antibody or the antibody fragment thereofaccording to any one of (1) to (18), which comprises complementaritydetermining regions (CDRs) of a heavy chain (H chain) variable region (Vregion) and light chain (L chain) V region of a monoclonal antibodyagainst CCR4, framework regions (FRs) of H chain V region and L chain Vregion of a human antibody, and H chain constant region (C region) and Lchain C region of a human antibody.

(20) The human CDR-grafted antibody or the antibody fragment thereofaccording to any one of (1) to (19), which comprises complementaritydetermining region (CDR) 1, CDR2 and CDR3 of an antibody heavy chain(1-1 chain) variable region (V region) having the amino acid sequencesrepresented by SEQ ID NOs:1, 2 and 3, respectively.

(21) The human CDR-grafted antibody or the antibody fragment thereofaccording to any one of (1) to (20), which comprises complementaritydetermining region (CDR) 1, CDR2 and CDR3 of an antibody light chain (Lchain) variable region (V region) having the amino acid sequencesrepresented by SEQ ID NOs:5, 6 and 7, respectively.

(22) The human CDR-grafted antibody or the antibody fragment thereofaccording to any one of (1) to (21), which comprises complementantydetermining region (CDR) 1, CDR2 and CDR3 of an antibody heavy chain (Hchain) variable region (V region) having the amino acid sequencesrepresented by SEQ ID NOs:1, 2 and 3, respectively, and complementaritydetermining region (CDR) 1, CDR2 and CDR3 of an antibody light chain (Lchain) V region having the amino acid sequences represented by SEQ IDNOs:5, 6 and 7, respectively.

(23) The human CDR-grafted antibody or the antibody fragment thereofaccording to any one of (1) to (22), which comprises an antibody heavychain (H chain) variable region (V region) comprising an amino acidsequence in which at least one amino acid residue selected from Ala atposition 40, Gly at position 42, Lys at position 43, Gly at position 44,Lys at position 76 and Ala at position 97 in the amino acid sequencerepresented by SEQ ID NO:4 is substituted with an other amino acid.

(24) The human CDR-grafted antibody or the antibody fragment thereofaccording to any one of (1) to (22), which comprises an antibody heavychain (H chain) variable region (V region) comprises an amino acidsequence in which at least one amino acid residue selected from Thr atposition 28 and Ala at position 97 in the amino acid sequencerepresented by SEQ ID NO:38 is substituted with an other amino acid.

(25) The human CDR-grafted antibody or the antibody fragment thereofaccording to any one of (1) to (24), which comprises an antibody lightchain (L chain) variable region (V region) comprising an amino acidsequence in which at least one amino acid residue selected from Ile atposition 2, Val at position 3, Gln at position 50 and Val at position 88in the amino acid sequence represented by SEQ ID NO:8 is substitutedwith an other amino acid.

(26) The human CDR-grafted antibody or the antibody fragment thereofaccording to any one of (1) to (23) and (25), which comprises anantibody heavy chain (H chain) variable region (V region) comprising anamino acid sequence in which at least one amino acid residue selectedfrom Ala at position 40, Gly at position 42, Lys at position 43, Gly atposition 44, Lys at position 76 and Ala at position 97 in the amino acidsequence represented by SEQ ID NO:4 is substituted with an other aminoacid residue; and an antibody light chain (L chain) V region comprisingan amino acid sequence in which at least one amino acid residue selectedfrom Ile at position 2, Val at position 3, Gln at position 50 and Val atposition 88 in the amino acid sequence represented by SEQ ID NO:8 issubstituted with an other amino acid residue.

(27) The human CDR-grafted antibody or the antibody fragment thereofaccording to any one of (1) to (22), (24) and (25), which comprises anantibody heavy chain (H chain) variable region (V region) comprising anamino acid sequence in which at least one amino acid residue selectedfrom Thr at position 28 and Ala at position 97 in the amino acidsequence represented by SEQ ID NO:38 is substituted with an other aminoacid residue; and an antibody light chain (L chain) V region comprisingan amino acid sequence in which at least one amino acid residue selectedfrom Ile at position 2, Val at position 3, Gln at position 50 and Val atposition 88 in the amino acid sequence represented by SEQ ID NO:8 issubstituted with an other amino acid residue.

(28) The human CDR-grafted antibody or the antibody fragment thereofaccording to any one of (1) to (22) and (25), which comprises anantibody heavy chain (H chain) variable region (V region) comprising theamino acid sequence represented by SEQ ID NO:4, 9, 10, 11, 38, 39, 40 or41.

(29) The human CDR-grafted antibody or the antibody fragment thereofaccording to any one of (1) to (24) and (28), which comprises anantibody light chain (L chain) variable region (V region) comprising theamino acid sequence represented by SEQ ID NO:8, 12, 13 or 14.

(30) The human CDR-grafted antibody or the antibody fragment thereofaccording to any one of (1) to (22), which comprises an antibody heavychain (H chain) variable region (V region) comprising the amino acidsequence represented by SEQ ID NO:4, 9, 10, 11, 38, 39, 40 or 41; and anantibody light chain (L chain) V region comprising the amino acidsequence represented by SEQ ID NO:8, 12, 13 or 14.

(31) The human CDR-grafted antibody or the antibody fragment thereofaccording to any one of (1) to (22), which comprises an antibody heavychain (H chain) variable region (V region) comprising the amino acidsequence represented by SEQ ID NO:9 or 10; and an antibody light chain(L chain) V region comprising the amino acid sequence represented by SEQID NO:14.

(32) The antibody fragment according to any one of (1) to (31), whereinthe antibody fragment is an antibody fragment selected from Fab, Fab′,F(ab′)₂, a single chain antibody (scFv), a dimerized variable region (Vregion) fragment (Diabody), a disulfide-stabilized V region fragment(dsFv) and a peptide comprising a complementarity determining region(CDR).

(33) A human CDR-grafted antibody, which is produced by a transformantKM8759 (FERM BP-8129) or KM8760 (FERM BP-8130), or the antibody fragmentthereof.

(34) A transformant which produces the human CDR-grafted antibody or theantibody fragment thereof according to any one of (1) to (33).

(35) The transformant according to (34), wherein the transformant isKM8759 (FERM BP-8129) or KM8760 (FERM BP-8130).

(36) A process for producing a transformant capable of producing thehuman CDR-grafted antibody or the antibody fragment thereof according toany one of (1) to (33), which comprises culturing the transformantaccording to (34) or (35) in a medium to form and accumulate the humanCDR-grafted antibody or the antibody fragment thereof in the culture;and recovering the antibody or the antibody fragment from the culture.

(37) A human CDR-grafted antibody or the antibody fragment thereof inwhich the human CDR-grafted antibody or the antibody fragment thereofaccording to any one of (1) to (33) is chemically or geneticallyconjugated with a radioisotope, a protein or an agent.

(38) A DNA which encodes the human CDR-grafted antibody or the antibodyfragment thereof according to any one of (1) to (33).

(39) A recombinant vector which comprises the DNA according to (38).

(40) A transformant which is obtainable by introducing the recombinantvector according to (39) into a host cell.

(41) A medicament which comprises at least one selected from the humanCDR-grafted antibody and the antibody fragment thereof according to anyone of (1) to (33) and (37) as an active ingredient.

(42) A therapeutic agent for treating CCR4-related diseases, whichcomprises at least one selected from the human CDR-grafted antibody andthe antibody fragment thereof according to any one of (1) to (33) and(37) as an active ingredient.

(43) The therapeutic agent according to (42), wherein the CCR4-relateddisease is a cancer or inflammatory diseases.

(44) The therapeutic agent according to (43), wherein the cancer is ablood cancer.

(45) The therapeutic agent according to (44), wherein the blood canceris leukemia or lymphomatosis.

(46) The therapeutic agent according to (43), wherein the inflammatorydisease is acute or chronic airway oversensitivity or bronchial asthma,atopic skin disease, allergic rhinitis or pollinosis.

(47) A diagnostic agent for CCR4-related diseases, which comprises atleast one selected from the human CDR-grafted antibody and the antibodyfragment thereof according to any one of (1) to (33) and (37) as anactive ingredient.

(48) The diagnostic agent according to (47), wherein the CCR4-relateddisease is a cancer or an inflammatory disease.

(49) The diagnostic agent according to (48), wherein the cancer is ablood cancer.

(50) The diagnostic agent according to (48), wherein the blood cancer isleukemia or lymphomatosis.

(51) The diagnostic agent according to (48), wherein the inflammatorydisease is chronic airway oversensitivity asthma, bronchial asthma,atopic skin disease, allergic rhinitis or pollinosis.

(52) A therapeutic agent for treating Th2-mediated immune diseases,which comprises at least one selected from the human CDR-graftedantibody and the antibody fragment thereof according to any one of (1)to (33) and (37) as an active ingredient.

(53) The therapeutic agent according to (52), wherein the Th2-mediatedimmune disease is chronic airway oversensitivity asthma, bronchialasthma, atopic skin disease, allergic rhinitis or pollinosis.

(54) A diagnostic agent for Th2-mediated immune diseases, whichcomprises at least one selected from the human CDR-grafted antibody andthe antibody fragment thereof according to any one of (1) to (33) and(37) as an active ingredient.

(55) The diagnostic agent according to (54), wherein the Th2-mediatedimmune disease is chronic airway oversensitivity asthma, bronchialasthma, atopic skin disease, allergic rhinitis or pollinosis.

(56) A method for immunologically detecting CCR4, which uses the humanCDR-grafted antibody and the antibody fragment thereof according to anyone of (1) to (33) and (37).

(57) A method for immunologically detecting a cell which expressed CCR4on the cell surface, which comprises using the human CDR-graftedantibody or the antibody fragment thereof according to any one of (1) to(33) and (37).

(58) A method for reducing or depleting a cell which expressed CCR4 onthe cell surface, which comprises using the human CDR-grafted antibodyor the antibody fragment thereof according to any one of (1) to (33) and(37).

(59) A method for inhibiting cytokine-production of a Th2 cell, whichcomprises using the human CDR-grafted antibody or the antibody fragmentthereof according to any one of (1) to (33) and (37).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows construction steps of a plasmid pKM2160Gal0.

FIG. 2 shows construction steps of a plasmid pKM2160Gal1. The symbol *shows the position of mutation genetic codon modifying an amino acidresidue.

FIG. 3 shows construction steps of a plasmid pKM2160Gal3. The symbol *shows the position of mutation genetic codon modifying an amino acidresidue.

FIG. 4 shows construction steps of a plasmid pKM2160LV0.

FIG. 5 shows construction steps of a plasmid pKM2160LV1. The symbol *shows the position of mutation genetic codon modifying an amino acidresidue.

FIG. 6 shows construction steps of a plasmid pKANTEX2160LV0 and plasmidpKANTEX2160Gal0LV0.

FIG. 7 shows reactivity of a culture supernatant obtained by transientlyexpressing an expression vector of each anti-CCR4 CDR-grafted antibodyin COS-7 cell, with a CCR4 partial peptide according to ELISA.

FIG. 8 shows reactivity of a culture supernatant obtained by transientlyexpressing an expression vector of each anti-CCR4 CDR-grafted antibodyprepared by using other FRs in COS 7 cell, with CCR4 partial peptideaccording to ELISA.

FIG. 9 shows reactivity of a purified anti-CCR4 CDR-grafted antibodywith a CCR4 partial peptide.

FIG. 10 shows reactivity of a purified anti-CCR4 CDR-grafted antibodywith a CCR4 high expression cell (CCR4/EL-4).

FIG. 11 shows affinity of a purified anti-CCR4 CDR-grafted antibody witha CCR4 partial peptide measured using a surface plasmon resonancesensor.

FIG. 12 shows cytotoxicity according to ADCC activity against CCR4/EL-4cell.

FIG. 13 shows effect on inhibition of production of IL-4, IL-13 andIFN-γ from human PBMC.

FIG. 14 shows binding activity of each antibody to a human platelet.

FIG. 15 shows construction steps of plasmids pKM2160VH41 andpKM2160VL61.

FIG. 16 shows construction step of a plasmid pKANTEX2160H.

FIG. 17 shows construction step of a plasmid pKANTEX2160.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, the CCR4-related diseases include cancers,inflammatory diseases and the like.

In the present invention, the cancers include blood cancers,particularly leukemia, lymphomatosis and the like.

In the present invention, the inflammatory diseases include acute orchronic airway oversensitivity or bronchial asthma; atopic skin diseasessuch as atopic dermatitis; allergic rhinitis; pollinosis; and the like.

In the present invention, the Th2-mediated immune diseases can be anyimmune diseases to which Th2 cell is related, and include acute orchronic airway oversensitivity or bronchial asthma; atopic skin diseasessuch as atopic dermatitis; allergic rhinitis; pollinosis; interstitialpneumonia; pulmonary fibrosis; autoimmune diseases such as systemiclupus erythematosus; and the like.

The human CDR-grafted antibody which specifically reacts with CCR4(anti-CCR4 CDR-grafted antibody) and an antibody fragment thereof of thepresent invention (hereinafter, both will be generally referred to asthe antibody of the present invention in some cases) are not limited, solong as it is a human CDR-grafted antibody which specifically reactswith the extracellular region of human CCR4 but does not react with ahuman platelet or an antibody fragment thereof. The term “does not reactwith a human platelet” means that the antibody shows substantially nobinding activity to a human platelet. Specifically, it means that thebinding activity is not shown when measured by a flow cytometer.

Also, the antibody of the present invention is an antibody whichspecifically reacts with the extracellular region of human CCR4 and hasa cytotoxic activity for CCR4-expressing cells.

The cytotoxic activity includes CDC activity and ADCC activity.

Also, the antibody of the present invention includes antibodies whichspecifically react with preferably a region comprising positions 1 to39, 98 to 112, 176 to 206 or 271 to 284 in the amino acid sequencerepresented by SEQ ID NO:48, more preferably a region comprisingpositions 2 to 29 in the amino acid sequence represented by SEQ ID NO:48(SEQ ID NO:36), still more preferably a region comprising positions 12to 29 in the amino acid sequence represented by SEQ ID NO:48 (SEQ IDNO:37), and most preferably a region comprising positions 12 to 25 inthe amino acid sequence represented by SEQ ID NO:48.

The human CDR-grafted antibody represents an antibody in which aminoacid sequences of CDR of VH and VL in an antibody derived from anon-human animal are grafted to appropriate positions of VH and VL of ahuman antibody.

The human CDR-grafted antibody of the present invention can be producedby constructing cDNAs encoding V regions in which amino acid sequencesof CDR of VH and VL in an antibody derived from a non-human animal,which specifically reacts with CCR4, are grafted to FR of VH and VL in ahuman antibody, inserting them respectively into an expression vectorfor animal cell having DNA encoding CH and H chain C region (hereinafterreferred to as “CL”) of a human antibody to construct a humanCDR-grafted antibody expression vector, and then introducing it into ananimal cell to express the human CDR-grafted antibody.

As the method for selecting FR amino acid sequences of VH and VL of ahuman antibody, any method can be used, so long as they are derived fromhuman antibodies. Examples include FR amino acid sequences of VH and VLin human antibodies registered in data bases such as Protein Data Bankand the like, or amino acid sequences common in each subgroup of FR ofVH and VL in human antibodies (Sequences of Proteins of ImmunologicalInterest, US Dep. Health and Human Services, 1991).

Any CH in the antibody of the present invention can be used, so long asit belongs to human immunoglobulin (hereinafter referred to as “hIg”).Preferably, an hIgG class, and any one of γ1, γ2, γ3 and γ4 subclassesbelonging to the hIgG class can be used. Also, any CL in the humanCDR-grafted antibody can be used, so long as it belongs to the hIg, andthose of κ class or λ class can be used.

The antibody of the present invention includes human CDR-graftedantibodies or the antibody fragments thereof which comprise antibody HVCDR1, CDR2 and CDR3 comprising the amino acid sequences represented bySEQ ID NOs: 1, 2 and 3, respectively, and/or VL CDR1, CDR2 and CDR3comprising the amino acid sequences represented by SEQ ID NOs:5, 6 and7, respectively.

Preferred examples include human CDR-grafted antibodies in which VH inthe antibody comprises the amino acid sequence represented by SEQ IDNO:4 or 38 and/or VL comprises the amino acid sequence represented bySEQ ID NO:8.

More preferable examples include:

a human CDR-grafted antibody which comprises VH of the antibodycomprising an amino acid sequence in which at least one amino acidresidue selected from Ala at position 40, Gly at position 42, Lys atposition 43, Gly at position 44, Lys at position 76 and Ala at position97 in the amino acid sequence represented by SEQ ID NO:4 is substitutedwith an other amino acid residue,

a human CDR-grafted antibody which comprises VH in the antibodycomprising an amino acid sequence in which at least one amino acidresidue selected from Thr at position 28 and Ala at position 97 in theamino acid sequence represented by SEQ ID NO:38 is substituted with another amino acid residue,

a human CDR-grafted antibody which comprises VL in the antibodycomprising an amino acid sequence in which at least one amino acidresidue selected from Ile at position 2, Val at position 3, Gln atposition 50 and Val at position 88 in the amino acid sequencerepresented by SEQ ID NO:8 is substituted with an amino acid residue.

a human CDR-grafted antibody which comprises VH in the antibodycomprising an amino acid sequence in which at least one amino acidresidue selected from Ala at position 40, Gly at position 42, Lys atposition 43, Gly at position 44, Lys at position 76 and Ala at position97 in the amino acid sequence represented by SEQ ID NO:4 is substitutedwith an other amino acid residue; and VL in the antibody comprising anamino acid sequence in which at least one amino acid residue selectedfrom Ile at position 2, Val at position 3, Gln at position 50 and Val atposition 88 in the amino acid sequence represented by SEQ ID NO:8 issubstituted with an other amino acid residue,

a human CDR-grafted antibody which comprises VH in the antibodycomprising an amino acid sequence in which at least one amino acidresidue selected from Thr at position 28 and Ala at position 97 in theamino acid sequence represented by SEQ ID NO:38 is substituted with another amino acid residue; and VL in the antibody VL comprising an aminoacid sequence in which at least one amino acid residue selected from Ileat position 2, Val at position 3, Gln at position 50 and Val at position88 in the amino acid sequence represented by SEQ ID NO:8 is substitutedwith an other amino acid residue,

and the like.

The present invention includes antibodies which comprise the amino acidsequence in which one or more amino acid is deleted, substituted,inserted or added and specifically react with CCR4 as described above,and the antibody fragments thereof.

In the present invention, one or more amino acid deletion, substitution,insertion or addition in the amino acid sequence means that one or moreamino acids are deleted, substituted, inserted and/or added to at one orplural positions in the amino acid sequence. The deletion, substitution,insertion and/or addition can be caused in the same amino acid sequencesimultaneously. Also, the amino acid residue substituted, inserted oradded can be natural or non-natural. The natural amino acid residueincludes L-alanine, L-asparagine, L-aspartic acid, L-glutamine,L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine,L-lysinc, L-methionine, L-phenylalanine, L-proline, L-serine,L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cysteine and thelike.

Thereinafter, preferred examples of amino acid residues which aresubstituted with each other are shown. The amino acid residues in thesame group can be substituted with each other.

Group A:

leucine, isoleucine, norleucine, valine, norvaline, alanine,2-aminobutanoic acid, methionine, O-methylsenne, t-butylglycinc,t-butylalanine, cyclohexylalanine;

Group B:

aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid,2-aminoadipic acid, 2-aminosuberic acid;

Group C:

asparagine, glutamine;

Group D:

lysine, arginine, ornithine, 2,4-diaminobutanoic acid,2,3-diaminopropionic acid;

Group E:

proline, 3-hydroxyproline, 4-hydroxyproline;

Group F:

serine, threonine, homoserine;

Group G:

phenylalanine, tyrosine.

The antibody fragment of the present invention includes Fab, Fab′,F(ab′)₂, scFv, Diabody, dsFv, a peptide comprising CDR, and the like.

An Fab is an antibody fragment having a molecular weight of about 50,000and antigen binding activity, in which about a half of the N-terminalside of H chain and the entire L chain, among fragments obtained bytreating IgG with a protease, papain (cut at an amino acid residue atposition 224 of the H chain), are bound together through a disulfidebond.

The Fab of the present invention can be obtained by treating a humanCDR-grafted antibody of the present invention which specifically reactswith CCR4, with a protease, papain. Also, the Fab can be produced byinserting DNA encoding Fab of the antibody into an expression vector forprokaryote or an expression vector for eukaryote, and introducing thevector into a prokaryote or eukaryote to express the Fab.

An F(ab′)₂ is an antibody fragment having a molecular weight of about100,000 and antigen binding activity, which is slightly larger than theFab bound via a disulfide bond of the hinge region, among fragmentsobtained by treating IgG with a protease, pepsin.

The F(ab′)₂ of the present invention can be obtained by treating a humanCDR-grafted antibody which specifically reacts with CCR4, with aprotease, pepsin. Also, the F(ab′)₂ can be produced by binding Fab′described below via a thioether bond or a disulfide bond.

An Fab′ is an antibody fragment having a molecular weight of about50,000 and antigen binding activity, which is obtained by cutting adisulfide bond of the hinge region of the F(ab′)₂.

The Fab′ of the present invention can be obtained by treating theF(ab′)₂ which specifically reacts with CCR4, with a reducing agent,dithiothreitol. Also, the Fab′ of the present invention can be producedby inserting DNA encoding an Fab′ of a human CDR-grafted antibody of thepresent invention which specifically reacts with CCR4 into an expressionvector for prokaryote or an expression vector for eukaryote, andintroducing the vector into a prokaryote or eukaryote to express theFab′.

An scFv is a VH-P-VL or VL-P-VH polypeptide in which one chain VH andone chain VL are linked using an appropriate peptide linker (P) of 12 ormore residues and which has an antigen-binding activity.

The scFv of the present invention can be produced by obtaining cDNAsencoding VH and VL of a human CDR-grafted antibody which specificallyreacts with CCR4 of the present invention, constructing DNA encodingscFv, inserting the DNA into an expression vector for prokaryote or anexpression vector for eukaryote, and then introducing the expressionvector into a prokaryote or eukaryote to express the scFv.

A diabody is an antibody fragment in which scFv's having the same ordifferent antigen binding specificity forms a dimer, and has an divalentantigen binding activity to the same antigen or two specific antigenbinding activity to different antigens.

The diabody of the present invention, for example, a divalent diabodywhich specifically reacts with CCR4, can be produced by obtaining cDNAsencoding VH and VL of an antibody which specifically reacts with CCR4,constructing DNA encoding scFv having a polypeptide linker of 3 to 10residues, inserting the DNA into an expression vector for prokaryote oran expression vector for eukaryote, and then introducing the expressionvector into a prokaryote or eukaryote to express the diabody.

A dsFV is obtained by binding polypeptides in which one amino acidresidue of each of VH and VL is substituted with a cysteine residue viaa disulfide bond between the cysteine residues. The amino acid residuewhich is substituted with a cysteine residue can be selected based on athree-dimensional structure estimation of the antibody in accordancewith the method shown by Reiter et al. (Protein Engineering, 7, 697(1994)).

The dsFv of the present invention can be produced by obtaining cDNAsencoding VH and VL of a human CDR-grafted antibody which specificallyreacts with CCR4 of the present invention, constructing DNA encodingdsFv, inserting the DNA into an expression vector for prokaryote or anexpression vector for eukaryote, and then introducing the expressionvector into a prokaryote or eukaryote to express the dsFv.

A peptide comprising CDR is constituted by including at least one regionof H chain and L chain CDRs. Plural CDRs can be bound directly or via anappropriate peptide linker.

The peptide comprising CDR of the present invention can be produced byobtaining cDNA encoding CDR of VH and VL of a human CDR-grafted antibodywhich specifically reacts with CCR4, constructing DNA encoding CDR,inserting the DNA into an expression vector for prokaryote or anexpression vector for eukaryote, and then by introducing the expressionvector into a prokaryote or eukaryote to express the peptide. Also, thepeptide comprising CDR can also be produced by a chemical synthesismethod such as an Fmoc method (fluorenylmethoxycarbonyl method), a tBocmethod (t-butyloxycarbonyl method), or the like.

The antibody of the present invention includes antibody derivatives inwhich a radioisotope, a protein, an agent or the like is chemically orgenetically conjugated to the antibody of the present invention.

The antibody derivatives of the present invention can be produced bychemically conjugating a radioisotope, a protein or a agent to theN-terminal side or C-terminal side of an H chain or an L chain of anantibody or antibody fragment which specifically reacts with CCR4, to anappropriate substituent group or side chain of the antibody or antibodyfragment or to a sugar chain in the antibody or antibody fragment(Antibody Engineering Handbook, edited by Osamu Kanemitsu, published byChijin Shokan (1994)).

Also, it can be genetically produced by linking a DNA encoding theantibody or the antibody fragment of the present invention whichspecifically reacts with CCR4 to other DNA encoding a protein to bebound, inserting the DNA into an expression vector, and introducing theexpression vector into a host cell.

The radioisotope includes ¹³¹I, ¹²⁵I and the like, and it can beconjugated to the antibody by, e.g., a chloramine T method.

The agent is preferably a low molecular weight compound. Examplesinclude anticancer agents such as alkylating agents (e.g., nitrogenmustard, cyclophosphamide), metabolic antagonists (e.g., 5-fluorouracil,methotrexate), antibiotics (e.g., daunomycin, bleomycin, mitomycin C,daunorubicin, doxorubicin), plant alkaloids (e.g., vincristine,vinblastine, vindesine), hormone drugs (e.g., tamoxifen, dexamethasone),and the like (Clinical Oncology, edited by Japanese Society of ClinicalOncology, published by Cancer and Chemotherapy (1996));anti-inflammatory agents such as steroid agents (e.g., hydrocortisone,prednisone), non-steroidal drugs (e.g., aspirin, indometacin),immunomodulators (e.g., aurothiomalate, penicillamine),immunosuppressing agents (e.g., cyclophosphamide, azathioprine) andantihistaminic agents (e.g., chlorpheniramine maleate, clemastine)(Inflammation and Anti-inflammatory Therapy, Ishiyaku Shuppan (1982));and the like. The method for conjugating daunomycin to an antibodyincludes a method in which daunomycin and an amino group of an antibodyare conjugated via glutaraldehyde, a method in which an amino group ofdaunomycin and a carboxyl group of an antibody are conjugated via awater-soluble carbodiimide, and the like.

The protein is preferably cytokine which activates immune cells.Examples include human interleukin 2 (hereinafter referred to as“hIL-2”), human granulocyte macrophage colony-stimulating factor(hereinafter referred to as “hGM-CSF”), human macrophagecolony-stimulating factor (hereinafter referred to as “hM-CSF”), humaninterleukin 12 (hereinafter referred to as “hIL-12”), and the like.Also, in order to inhibit cancer cells directly, a toxin such as ricin,diphtheria toxin and the like, can be used. For example, a fusionantibody with a protein can be produced by linking a cDNA encoding anantibody or antibody fragment to other cDNA encoding the protein,constructing DNA encoding the fusion antibody, inserting the DNA into anexpression vector for prokaryote or an expression vector for eukaryote,and then introducing it into a prokaryote or eukaryote to express thefusion antibody.

Methods for producing the human CDR-grafted antibody and the antibodyfragment thereof which specifically react with CCR4, methods forevaluating the activity thereof and methods for using them are explainedbelow.

1. Preparation of Human CDR-Grafted Antibody

(1) Construction of Humanized Antibody Expression Vector

A humanized antibody expression vector is an expression vector foranimal cell into which genes encoding CH and CL of a human antibody havebeen inserted, and is constructed by cloning each of CH and CL of ahuman antibody into an expression vector for animal cell.

The C region of a human antibody can be CH and CL of any human antibody.Examples include CH of γ subclass and CL of κ class of a human antibody,and the like. Also, cDNA can be used. As the expression vector foranimal cell, any expression vector can be used, so long as a C region ofa human antibody can be inserted and expressed. Examples include pAGE107(Cytotechnology, 3, 133 (1990)), PAGE 103 (J. Biochem., 101, 1307(1987)), pHSG274 (Gene, 27, 223 (1984)), pKCR (Proc Natl Acad. Sci. USA,78, 1527 (1981)), pSGIβd2-4 (Cytotechnology, 4, 173 (1990)),pSE1UK1Sed1-3 (Cytotechnol, 13, 79 1993)) and the like. A promoter andenhancer used for an expression vector for animal cell includes an SV40early promoter and enhancer (J. Biochem., 101, 1307 (1987)), a Moloneymouse leukemia virus LTR promoter and enhancer (Biochem. Biophys. Res.Comm., 149, 960 (1987)), an immunoglobulin H chain promoter (Cell, 41,479 (1985)) and enhancer (Cell, 33, 717 (1983)), and the like.

The humanized antibody expression vector can be either of a type inwhich a gene encoding an antibody H chain and a gene encoding anantibody L chain exist on separate vectors or of a type in which bothgenes exist on the same vector (tandem type). In respect of easiness ofconstruction of a humanized antibody expression vector, easiness ofintroduction into animal cells, and balance between the expressionamounts of antibody H and L chains in animal cells, a tandem type of thehumanized antibody expression vector is more preferred (J. Immunol.Methods, 167, 271 (1994)). The tandem type of the humanized antibodyexpression vector includes pKANTEX93 (WO 97/10354), pEE18 (HYBRIDOMA,17, 559 (1998)) and the like.

The constructed humanized antibody expression vector can be used forexpression of a human CDR-grafted antibody in animal cells.

(2) Construction of cDNA Encoding V Region of Human CDR-Grafted Antibody

cDNAs encoding VH and VL of a human CDR-grafted antibody can be obtainedas follows. First, amino acid sequences of FRs in VH and VL of a humanantibody to which amino acid sequences of CDRs in VH and VL of anantibody derived from a non-human animal antibody are grafted areselected. Any amino acid sequences of FRs in VH and VL of a humanantibody can be used, so long as they are derived from human. Examplesinclude amino acid sequences of FRs in VH and VL of human antibodiesregistered in database such as Protein Data Bank and the like, and aminoacid sequences common to subgroups of FRs in VH and VL of humanantibodies (Sequences of Proteins of Immunological Interest, US Dept.Health and Human Services (1991)), and the like. In order to produce ahuman CDR-grafted antibody having potent activity, amino acid sequenceshaving high homology (at least 60% or more) with amino acid sequence ofFRs in VH and VL of a target antibody derived from a non-human animal ispreferably selected.

Then, amino acid sequences of CDRs in VH and VL of the antibody derivedfrom a non-human animal are grafted to the selected amino acid sequencesof FRs in VH and VL of a human antibody to design amino acid sequencesof VH and VL of a human CDR-grafted antibody. The designed amino acidsequences are converted to DNA sequences by considering the frequency ofcodon usage found in nucleotide sequences of genes of antibodies(Sequence of Proteins of Immunological Interest, US Dept. Health andHuman Services (1991)), and the DNA sequences encoding the amino acidsequences of VH and VL of a human CDR-grafted antibody are designed.Several synthetic DNAs having a length of about 100 to 200 nucleotidesare synthesized, and PCR is carried out using them. In this case, it ispreferred in each of VH and VL that 4 or 6 synthetic DNAs are designedin view of the reaction efficiency of PCR and the lengths of DNAs whichcan be synthesized. Furthermore, they can be easily cloned into thehumanized antibody expression vector constructed in the item 1(1) byintroducing the recognition sequence of an appropriate restrictionenzyme to the 5′ end of the synthetic DNAs present on the both ends.After the PCR, an amplified product is cloned into a plasmid such aspBluescript SK (−) (manufactured by Stratagene) or the like, and thenucleotide sequences are determined to obtain a plasmid having DNAsequences encoding VH and VL of a designed human CDR-grafted antibody.

(3) Modification of Amino Acid Sequence of V Region of Human CDR-GraftedAntibody

It is known that when a human CDR-grafted antibody is produced by simplygrafting only CDRs in VH and VL of an antibody derived from a non-humananimal into FRs in VH and VL of a human antibody, its antigen-bindingactivity is lower than that of the original antibody derived from anon-human animal (BIO/TECHNOLOGY, 9, 266 (1991)). As the reason, it isconsidered that several amino acid residues in not only CDRs but alsoFRs directly or indirectly relate to antigen-binding activity in VH andVL of the original antibody derived from a non-human animal, and thatthey axe changed to different amino acid residues of different FRs in VHand VL of a human antibody. In order to solve the problem, in humanCDR-grafted antibodies, among the amino acid sequences of FRs in VH andVL of a human antibody, an amino acid residue which directly relates tobinding to an antigen, or an amino acid residue which indirectly relatesto binding to an antigen by interacting with an amino acid residue inCDR or by maintaining the three-dimensional structure of an antibody isidentified and modified to an amino acid residue which is found in theoriginal non-human animal antibody to thereby increase the antigenbinding activity which has been decreased (BIO/TECHNOLOGY, 9, 266(1991)). In the production of a human CDR-grafted antibody, how toefficiently identify the amino acid residues relating to the antigenbinding activity in FR is most important, so that the three-dimensionalstructure of an antibody is constructed and analyzed by X-raycrystallography (J. Mol. Biol., 112, 535 (1977)), computer-modeling(Protein Engineering, 7, 1501 (1994)) or the like. Although theinformation of the three-dimensional structure of antibodies has beenuseful in the production of a human CDR-grafted antibody, no method forproducing a human CDR-grafted antibody which can be applied to anyantibodies has been established yet. Therefore, various attempts must becurrently be necessary, for example, several modified antibodies of eachantibody are produced and the relationship between each of the modifiedantibodies and its antibody binding activity is examined.

The modification of the selected amino acid sequence of FRs in VH and VLof a human antibody can be accomplished using various synthetic DNA formodification according to PCR. With regard to the amplified productobtained by the PCR, the nucleotide sequence is determined according tothe method as described in the item 1(2) so that whether the objectivemodification has been carried out is confirmed.

(4) Construction of Human CDR-Grafted Antibody Expression Vector

A human CDR-grafted antibody expression vector can be constructed bycloning cDNAs encoding VH and VL of the human CDR-grafted antibodyconstructed in the items 1(2) and 1(3) into upstream of the genesencoding VH and VL of the human antibody in the humanized antibodyexpression vector as described in the item 1(1). For example, whenrecognition sites for an appropriate restriction enzymes are introducedto the 5′-terminal of synthetic DNAs positioned at both ends amongsynthetic DNAs used in the construction of VH and VL of the humanCDR-grafted antibody in the items 1(2) and (3), cloning can be carriedout so that they are expressed in an appropriate form in upstream ofgenes encoding CH and CL of the human antibody in the humanized antibodyexpression vector as described in the item 1(1).

(5) Transient Expression of Human CDR-Grafted Antibody

In order to efficiently evaluate the antigen binding activity of varioushuman CDR-grafted antibodies produced, the human CDR-grafted antibodiescan be expressed transiently using the human CDR-grafted antibodyexpression vector as described in the item 1(4) or the modifiedexpression vector thereof. Any cell can be used as a host cell, so longas the host cell can express a human CDR-grafted antibody. Generally,COS-7 cell (ATCC CRL1651) is used in view of its high expression amount(Methods in Nucleic Acids Res., CRC Press, p. 283 (1991)). The methodfor introducing the expression vector into COS-7 cell includes aDEAE-dextran method (Methods in Nucleic Acids Res., CRC Press, p. 283(1991)), a lipofection method (Proc. Natl. Acad. Sci. USA, 84, 7413(1987)), and the like.

After introduction of the expression vector, the expression amount andantigen binding activity of the human CDR-grafted antibody in theculture supernatant can be determined by the enzyme immunoassay (ELISA);Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter14 (1988), Monoclonal Antibodies Principles and Practice, Academic PressLimited (1996)) and the like.

(6) Stable Expression of Human CDR-Grafted Antibody

A transformant which produces a human CDR-grafted antibody stably can beobtained by introducing into an appropriate host cell the humanCDR-grafted antibody expression vector described in the item 1(4).

The method for introducing the expression vector into a host cellincludes electroporation (Cytotechnology, 3, 133 (1990)) and the like.

Any cell can be used as the host cell into which the human CDR-graftedantibody expression vector is to be introduced, so long as it canexpress a human CDR-grafted antibody. Examples include mouse SP2/0-Ag14cell (ATCC CRL1581), mouse P3X63-Ag8.653 cell (ATCC CRL1580), CHO cellin which a dihydrofolate reductase (defr) gene is detective (Proc. Natl.Acad. Sci. USA., 77, 4216 (1980)), rat YB2/3HL.P2.G11.16Ag.20 cell(YB2/0 cell; ATCC CRL1662) and the like.

After introduction of the expression vector, transformants which expressa human CDR-grafted antibody stably are selected by culturing in amedium for animal cell culture containing an agent such as G418 sulfate(G418; manufactured by Sigma) or the like (J. Immuol. Methods, 167, 271(1994)). The medium for animal cell culture includes PRMI1640 medium(manufactured by Nissui Pharmaceutical), GIT medium (manufactured byNissui Pharmaceutical), EX-CELL302 medium (manufactured by JRH), IMDMmedium (manufactured by GIBCO BRL), Hybridoma-SFM medium (manufacturedby GIBCO BRL), media obtained by adding various additives such as FBS tothese media, and the like. The human CDR-grafted antibody can beproduced and accumulated in a culture medium by culturing the selectedtransformants in a medium. The expression amount and antigen bindingactivity of the humanized antibody in the culture supernatant can bemeasured by ELISA or the like. Also, in the transformant, the expressionamount of the human CDR-grafted antibody can be increased by using dhframplification system or the like (J. Immuol. Methods, 167, 271 (1994)).

The human CDR-grafted antibody can be purified from the culturesupernatant of the transformant by using a protein A column (Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter 8 (1988),Monoclonal Antibodies: Principles and Practice, Academic Press Limited(1996)). Any other conventional methods for protein purification can beused. For example, the humanized antibody can be purified by acombination of gel filtration, ion-exchange chromatography,ultrafiltration and the like. The molecular weight of the H chain or theL chain of the purified humanized antibody or the antibody molecule as awhole is determined by polyacrylamide gel electrophoresis (SDS-PAGE;Nature, 227, 680 (1970)), Western blotting (Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, Chapter 12 (1988), MonoclonalAntibodies: Principles and Practice, Academic Press Limited (1996)) andthe like.

2. Preparation of Antibody Fragment

The antibody fragment can be prepared based on the humanized antibodydescribed in the item I using genetic engineering or proteinengineering. The antibody fragment includes Fab, F(ab′)₂, Fab′, sdFv,diabody, dsFv, a peptide comprising CDR, and the like.

(1) Preparation of Fab

Fab can be prepared by treating IgG with a proteolytic enzyme papain.After the papain treatment, when the original antibody is an IgGsubclass having a protein A binding activity, uniform Fab can berecovered by separating it from IgG molecules and Fc fragments bypassing through a protein A column (Monoclonal Antibodies: Principlesand Practice, third edition (1995)). When the original antibody is anantibody of IgG subclass having no protein A binding activity, Fab canbe recovered by ion exchange chromatography in a fraction eluted at alow salt concentration (Monoclonal Antibodies: Principles and Practice,third edition (1995)). In addition, Fab can also be prepared by geneticengineering techniques using Escherichia coli. For example, an Fabexpression vector can be prepared by cloning the DNA encoding theantibody V region described in the items 1(2) and 1(3) into a vector forFab expression. As the vector for Fab expression, any vector can beused, so long as a DNA for Fab can be inserted and expressed. Examplesinclude pIT106 (Science, 240, 1041 (1988)) and the like. Fab can beformed and accumulated in an inclusion body or periplasmic space byintroducing the Fab expression vector into an appropriate Escherichiacoli. Active Fab can be obtained from the inclusion body by a refoldingmethod generally used for protein, and when it is expressed in theperiplasmic space, active Fab is leaked in the culture supernatant.Uniform Fab can be purified after the refolding or from the culturesupernatant using an antibody-linked column (Antibody Engineering, APractical Guide, W.H. Freeman and Company (1992)).

(2) Preparation of F(ab′)₂

F(ab′)₂ can be prepared by treating IgG with a proteolytic enzymepapain. After the papain treatment, it can be recovered as uniformF(ab′)₂ by a purification procedure similar to the case of Fab(Monoclonal Antibodies: Principles and Practice, third edition, AcademicPress (1995)). In addition, it can also be prepared by the methoddescribed in the item 2(3) in which Fab′ is treated with maleimide suchas o-PDM, bismaleimide hexane or the like to form a thioether bond, or amethod in which it is treated with DTNB to form an S-S bond (AntibodyEngineering, A Practical Approach, IRL PRESS (1996)).

(3) Preparation of Fab′

Fab′ can be prepared by genetic engineering techniques using Escherichiacoli. For example, an Fab′ expression vector can be constructed bycloning the DNA encoding the antibody V region described in the items1(2) and (3) into a vector for Fab′ expression. As the vector for Fab′expression, any vector can be used, so long as a DNA for Fab′ can beinserted and expressed. Examples include pAK19 (Bio/Technology, 10, 163(1992)) and the like. Fab′ can be formed and accumulated in an inclusionbody or periplasmic space by introducing the Fab′ expression vector intoan appropriate Escherichia coli. Active Fab′ can be obtained from theinclusion body by a refolding method generally used for protein, andwhen it is expressed in the periplasmic space, it can be recovered intoextracellular moiety by disrupting the cells with a treatment such aslysozyme partial digestion, osmotic pressure shock, sonication or thelike. Uniform Fab′ can be purified after the refolding or from thedisrupted cell suspension using a protein G column or the like (AntibodyEngineering, A Practical Approach, IRL PRESS (1996)).

(4) Preparation of scFv

scFv can be prepared using a phage or Escherichia coli by geneticengineering techniques. For example, a DNA encoding scFv is produced byligating DNAs encoding the antibody VH and VL described in the items1(2) and (3) via a DNA encoding a polypeptide linker comprising an aminoacid sequence of 12 residues or more. An scFv expression vector can beconstructed by cloning the resulting DNA into a vector for scFvexpression. As the vector for scFv expression, any vector can be used,so long as a DNA for scFv can be inserted and expressed. Examplesinclude pCANTAB5E (manufactured by Pharmacia), Phfa (Hum. AntibodyHybridoma, 5, 48 (1994)) and the like. The scFv expression vector wasintroduced into an appropriate Escherichia coli and infected with ahelper phage to thereby obtain a phage which expresses scFv on the phagesurface in a fused form with the phage surface protein. Also, scFv canbe formed and accumulated in the inclusion body or periplasmic space ofEscherichia coli into which scFv expression vector is introduced. ActivescFv can be obtained from the inclusion body by a refolding methodgenerally used for protein, and when it is expressed in the periplasmicspace, it can be recovered extracellularly by disrupting the cells witha treatment such as lysozyme partial digestion, osmotic pressure shock,sonication or the like. Uniform scFv can be purified after the refoldingor from the disrupted cell suspension by cation exchange chromatographyor the like (Antibody Engineering, A Practical Approach, IRL PRESS(1996)).

(5) Preparation of Diabody

Diabody can be prepared by changing the size of the polypeptide linkerfor preparing scFv to about 3 to 10 residues. A divalent diabody can beprepared when VH and VL of one antibody species is used, and a diabodyhaving two different specificity when VH and VL of two antibody speciesare used (FEBS Letters, 453, 164 (1999), Int. J. Cancer, 77, 763(1998)).

(6) Preparation of dsFv

dsFv can be prepared using Escherichia coli by genetic engineeringtechniques. First, DNAs in which an encoded amino acid residue isreplaced with a cysteine residue are produced by introducing mutationinto appropriate positions of the DNAs encoding the antibody VH and VLdescribed in the items 1(2) and 1(3). VH and VL expression vectors canbe produced by cloning each of the resulting DNAs into a vector for dsFvexpression. As the vector for dsFv expression, any vector can be used,so long as a DNA for dsFv can be inserted and expressed. Examplesinclude pULI9 (Protein Engineering, 7, 697 (1994)) and the like. The VHand VL expression vectors are introduced into an appropriate Escherichiacoli to thereby form and accumulate the VH and VL in the inclusion bodyor periplasmic space. The VH and VL are obtained from the inclusion bodyor periplasmic space and mixed, and active dsFv can be obtained by arefolding method generally used for protein. After the refolding, it canbe further purified by ion exchange chromatography and gel filtration orthe like (Protein Engineering, 7, 697 (1994)).

(7) Preparation of Peptide Comprising CDR

A peptide comprising CDR can be prepared by a chemical synthesis methodsuch as Fmoc, tBoc or the like. Also, a DNA encoding a peptidecomprising CDR is prepared, and the resulting DNA is cloned into anappropriate vector for expression to thereby prepare the peptidecomprising CDR. As the vector for expression, any vector can be used, solong as a DNA encoding a peptide comprising CDR is inserted andexpressed. Examples include pLEX (manufactured by Invitrogen), pAX4a+(manufactured by Invitrogen) and the like. The expression vector isintroduced into an appropriate Escherichia coli so that the peptidecomprising CDR can be formed and accumulated in the inclusion body orperiplasmic space. The peptide comprising CDR can be obtained from theinclusion body or periplasmic space, and it can be purified by ionexchange chromatography and gel filtration or the like (ProteinEngineering, 7, 697 (1994)).

3. Evaluation of Activity and Property of the Antibody of the PresentInvention

(1) Evaluation of Binding Activity for Antigen

A binding activity of the antibody of the present invention for anantigen can be measured by enzyme-linked immunosorbent assay (ELISA),fluorescent antibody technique (Cancer Immunol. Immunother., 36, 373(1993), surface plasmon resonance using such as BIAcore™ and the like.Specifically, a synthetic peptide comprising a CCR4 partial sequence isproduced and a conjugate is prepared by chemically linking it to acarrier protein such as bovine serum albumin or the like. A CCR4 bindingactivity of the antibody of the present invention can be measured byimmobilizing the conjugate on an ELISA plate, allowing it to react withthe antibody of the present invention, further allowing it to react witha labeled antibody or binding fragment such as a peroxidase- orbiotin-labeled antibody or binding fragment, and then measuring acoloring dye using an absorption photometer.

(2) Reactivity for CCR4-Expressing Cells

In order to examine the reactivity for CCR4-expressing cells, it ispreferable to use a method for efficiently detecting CCR4 expressed onthe cell surface. The method includes flow cytometry using fluorescentantibody technique and the like. In addition, when reactivity withliving body cells such as platelet and the like are examined, it ispreferable to carry out the examination under conditions as close aspossible to the living body. Because of these reasons, when reactivityof the anti-CCR4 antibody of the present invention is examined, it ismost desirable to carry out the examination by flow cytometry usingfluorescent antibody technique.

The antibody used in the fluorescent antibody technique may be either anantibody labeled with a fluorescent material such as FITC, biotin or thelike, or an unlabeled antibody. Depending on the presence or absence ofa label of the used antibody and its kind, fluorescence-labeled avidin,a fluorescence-labeled anti-human immunoglobulin antibody and the likeare used. The reactivity can be evaluated by carrying out the reactionby adding a sufficient amount of an anti-CCR4 antibody (generally from0.1 to 10 μg/ml as the final concentration) to a tested sample and bycomparing its reactivity with those of a negative control antibody and apositive control antibody.

(3) Cytotoxic Activity

The cytotoxic activity for CCR4-expressing cells can be evaluated bymeasuring CDC activity, ADCC activity and the like (Cancer Immunol.Immunother., 36, 373 (1993)). Changes in the amount of produced cytokinecan be measured by ELISA method, fluorescent antibody technique and thelike using an antibody for cytokine.

(4) Activity of Inhibiting Ligand Binding

An activity of inhibiting ligand binding of the anti-CCR4 antibody ofthe present invention can be examined by using a label of TARC or MDC asa ligand having a binding activity to CCR4 and a CCR4-expressing cell ora cell membrane fraction thereof. TARC or MDC can be labeled by anytechnique which can be detected, and examples include fluorescencelabeling, enzyme labeling, radiation labeling and the like. Specificexamples include the method by measuring the binding inhibition activityusing a radiation label described in WO 00/42074.

Also, an activity of inhibiting ligand binding of the anti-CCR4 antibodyof the present invention can be examined by using a cell responseinduced by binding of a ligand to CCR4 as an index. The cell responsemay be any type, so long as it is induced by contacting a ligand with aCCR4-expressing cell, and examples include changes in intracellularcalcium concentration, cell migration and the like. Specific examplesinclude the method measuring migration inhibition of a CCR4-expressingcell induced by a CCR4 ligand described in WO 00/42074.

(5) Examination of Recognition Sequence

An amino acid sequence recognizable by the antibody of the presentinvention can be determined by using a synthetic peptide designed basedon the primary sequence of its corresponding antigen protein.

A primary sequence of the synthetic peptide is designed based on theprimary sequence of the antigen protein. In order to prepare a proteinin which the synthetic peptide is crosslinked with a carrier protein, acysteine residue can be added to the carboxyl terminal or amino terminalof the synthetic peptide. The resulting protein can be used in the ELISAwhich will be described later. Also, if necessary, the N-terminal andC-terminal of the synthetic peptide can be acetylated and amidated,respectively.

A peptide can be synthesized by a general liquid phase or solid phasepeptide synthesizing method, an any combined method thereof or amodified method thereof (International Journal of Peptide ProteinResearch, 35, 161-214 (1990), “Solid-Phase Peptide Synthesis”, Methodsin Enzymology, vol. 289, edited by Gregg B. Fields, Academic Press(1997), “Peptide Synthesis Protocols”, Methods in Molecular Biology,vol. 35, edited by Michael W. Pennington and Ben M. Dunn, Humana Press(1994)).

In addition, an automatic peptide synthesizer can also be used.Synthesis of a peptide by a peptide synthesizer can be carried out by acommercially available peptide synthesizer such as the peptidesynthesizer manufactured by Shimadzu Corp., the peptide synthesizermanufactured by Advanced ChemTech Inc, USA (hereinafter referred to as“ACT”) or the like, using N^(α)-Fmoc-amino acids, N^(α)-Boc-amino acidsor the like whose side chains are appropriately protected and inaccordance with respective synthesis programs. The protected amino acidsused as the material and carrier resins can be purchased from ABI Inc.,Shimadzu Corp., Kokusan Kagaku K.K., NovaBiochem, Watanabe Kagaku K.K.,ACT, AnaSpec Inc., Peptide Research Institute and the like.

As the method for determining an amino acid sequence recognizable by theantibody of the present invention using the synthetic peptide, anytechnique can be used, so long as it is a method which can detectbinding of the synthetic peptide to the antibody. For example, the aminoacid sequence recognizable by the antibody can be determined by labelingthe synthetic peptide with a fluorescent material, a radioactivematerial or the like and examining the binding activity of the resultinglabeled peptide to the antibody. Also, it can be carried out bycrosslinking the synthetic peptide with a protein such as bovine serumalbumin (BSA) or the like and evaluating the reactivity of the resultingprotein with the antibody by ELISA or the like. In addition, an aminoacid sequence recognizable by the antibody of the present invention canalso be determined by using a substance already confirmed that theantibody links thereto, such as an antibody protein, and examining asynthetic peptide which inhibits linking of the antibody to thesubstance.

4. Method for Detecting and Quantifying CCR4 Using Anti-CCR4-Antibody

The present invention relates to a method for immunologically detectingand determining CCR4 or a cell expressing CCR4 on the surface thereofusing the antibody of the present invention.

The methods for immunologically detecting and determining CCR4 or a cellexpressing CCR4 on the surface thereof using the antibody of the presentinvention include an immunofluorescent method, an enzyme-linkedimmunosorbent assay (ELISA), a radioactive material labeled immunoassay(RIA), an immunohitsochemical staining method such as an immunocytestaining method, an immunotissue staining method, or the like (ABCmethod, CSA method, etc.), the above enzyme immunoassay, a sandwichELISA (Monoclonal Antibody Experiment Manual (published by KodanshaScientific, 1987), Second Series Biochemical Experiment Course, Vol, 5,Immunobiochemistry Research Method, published by Tokyo Kagaku Dojin(1986)).

The immunofluorescent method comprises reacting a separated cell,tissue, or the like with the antibody of the present invention, reactingthe reactant with an anti-immunoglobulin antibody or binding fragmentlabeled with a fluorescence substance such as fluorescein isothiocyanate(FITC) or the like, and then measuring the fluorescence substance with aflow cytometer.

The enzyme-linked immunosorbent assay (ELISA) comprises reacting aseparated cell or cell lysate thereof, tissue or tissue lysate thereof,cell culture supernatant, serum, preural fluid, ascites fluid, ocularfluid or the like with the antibody of the present invention, reactingthe reactant with an anti-immunoglobulin antibody or binding fragmentlabeled with an enzyme such as peroxydase, biotin, or the like, and thenmeasuring the resultant developed dye with an absorption photometer.

The radioactive material labeled immunoassay (RIA) comprises reacting aseparated cell or cell lysate, tissue or tissue lysate, cell culturesupernatant, serum, preural fluid, ascites fluid, ocular fluid or thelike with the antibody of the present invention, further reacting thereactant with an anti-immunoglobulin antibody or binding fragmentlabeled with radioisotope, and then measuring the radioactivity with ascintillation counter or the like.

The immunocyte staining and immunotissue staining methods comprisereacting a separated cell, tissue or the like with the antibody of thepresent invention, reacting the reactant with an anti-immunoglobulinantibody or binding fragment labeled with a fluorescence substance suchas fluorescein isothiocyanate (FITC) or the like, or an enzyme such asperoxydase, biotin or the like, and then observing the cell, tissue orthe like with a microscope.

The sandwich ELISA is a method which comprises adsorbing, on a plate,one of two antibodies having a different epitope among the antibodies ofthe present invention; labeling another antibody with a fluorescencesubstance such as FITC or the like, or an enzyme such as peroxydase,biotin or the like; reacting a separated cell or cell lysate, tissue ortissue lysate, cell culture supernatant, scrum, preural fluid, ascitesfluid, ocular fluid, or the like with the antibody-adsorbing plate; andthen reacting it with the labeled antibody for carrying out a reactionaccording to the labeled substance.

5. Method for Using Human CDR-Grafted Antibody or Antibody FragmentThereof.

Since the antibody of the present invention specifically binds to CCR4which is expressed on a cultured cell line and shows cytotoxic activitysuch as CDC activity, ADCC activity and the like, it will be useful indiagnosing and treating diseases relating to CCR4 such as Th2-mediateddiseases and the like. Also, since the proportion of amino acidsequences derived from human antibody is higher than that in antibodiesof a non-human animal, it is expected that it shows strong cytotoxicactivity in the human body, it does not show immunogenicity, and itseffects continue for a king time.

In addition, the production of Th2 cytokines which are produced by cellssuch as IL-4, IL-5, IL-13 and the like, can be inhibited byadministering the antibody of the present invention to cells or tissuesof an experimental subject.

As the cell expressing CCR4 relating to the present invention, Th2 celland the like are exemplified. The Th2 cell used in the present inventionis preferably activated Th2 cell or memory Th2 cell. Examples includecells having CD45RA− or CD45RO+ and CD4+ properties.

The cytotoxic activities of the antibody of the present invention aregenerated, e.g., when the antibody of the present invention binds toCCR4-expressing cells such as a Th2 cell to thereby induce apoptosis inthe cell. Also, the cell can be obstructed and depleted by inducingapoptosis.

Also, the method for diagnosing Th2-mediated immune diseases or cancersincludes a method in which a human CCR4 positive cell existing in cellsor tissues of an experimental subject is immunologically detected asdescribed above.

Furthermore, the antibody of the present invention can be used as adiagnostic agent for CCR4-related diseases such as Th2-mediated immunediseases or cancers, or diseases in which the morbid states advance dueto abnormal increase or decrease of Th2 cells.

Moreover, since the antibody of the present invention can reduce ordeplete CCR4-expressing cells by its cytotoxic activity, it can providea diagnostic method or therapeutic method for CCR-4-related diseasessuch as Th2-mediated immune diseases or cancers, which uses the antibodyof the present invention, and therapeutic and preventive agents forCCR4-related diseases such as Th2-mediated immune diseases or cancerswhich comprises the antibody of the present invention as an activeingredient.

The Th2-mediated immune diseases include, irrespective of mild orsevere, inflammatory diseases such as acute or chronic airwayhypersensitivity or bronchial asthma, atopic skin diseases includingatopic dermatitis, allergic rhinitis, pollinosis, and the like; diseasescaused by inflammation competent cells such as eosinophil, mast cell andthe like which can be propagated or activated by cytokine and chemokinereleased from Th2 cells, biologically functional molecules such as IgEand the like which are produced by cytokine and chemokine released fromTh2 cells, and the like; and immune diseases in which the morbid statesadvance due to abnormal changes in Th2 cells.

The antibody of the present invention can be administered alone, but itis generally preferred to provide it in the form of a pharmaceuticalformulation produced by mixing it with at least one pharmaceuticallyacceptable carrier in accordance with a method well known in thetechnical field of pharmaceutics.

It is preferred to select a route of administration which is the mosteffective in carrying out the intended treatment such as oraladministration or parenteral administration, e.g., intraoraladministration, tracheal administration, rectal administration,subcutaneous injection, intramuscular injection, intravenous injection,and the like. Intravenous injection is preferred in an antibody orpeptide formulation.

The dosage form includes sprays, capsules, tablets, granules, syrups,emulsions, suppositories, injections, ointments, tapes, and the like.

Formulations suitable for oral administration include emulsions, syrups,capsules, tablets, powders, granules, and the like.

Liquid preparations such as emulsions and syrups, can be produced usingadditives such as water; saccharides, e.g., sucrose, sorbitol, fructose;glycols, e.g., polyethylene glycol, propylene glycol; oils, e.g., sesameoil, olive oil, soybean oil; antiseptics, e.g., p-hydroxybenzoate; andflavors, e.g., strawberry flavor, peppermint.

Capsules, tablets, powders, granules and the like can be produced usingadditives such as fillers, e.g., lactose, glucose, sucrose, mannitol;disintegrating agents, e.g., starch, sodium alginate; lubricants, e.g.,magnesium stearate; talc; binders, e.g., polyvinyl alcohol,hydroxypropylcellulose, gelatin; surfactants, e.g., fatty acid esters;and plasticizers, e.g., glycerine.

Formulations suitable for parenteral administration include injections,suppositories, sprays, and the like.

Injections can be prepared using a carrier such as a salt solution,glucose solution or a mixture thereof, or the like.

Suppositories can be prepared using a carrier such as cacao butter,hydrogenated fat, a carboxylic acid, or the like.

Also, sprays can be prepared from the antibody itself or using a carrieror the like which does not stimulate oral and airway mucous membranes ofpatients and can facilitate absorption of the antibody or antibodyfragment thereof by dispersing it as minute particles.

The carrier includes lactose, glycerine, and the like. Depending on theproperties of the antibody or peptide and the carrier to be used,aerosols, dry powders and the like can be produced. The additivesexemplified in the oral preparations can also be added to the parenteralpreparations.

The dose and frequency of administration vary depending on intendedtherapeutic effect, administration method, treating period, age, bodyweight and the like, but the dose is generally from 0.01 mg/kg to 20mg/kg per day per adult.

As discussed above, according to the present invention, a recombinantantibody and an antibody fragment thereof, which binds specifically tohuman CCR4 and contains novel CDRs for CCR4, are provided. The antibodyof the present invention is useful for the diagnosis or treatment ofCCR4-related diseases. Specifically, it is useful for the immunologicaldetection of a human Th2 cell by immunocyte staining and for thediagnosis or treatment of all Th2-mediated immune diseases includingbronchial asthma and atopic skin diseases, diseases in which the morbidstates advance due to abnormal balance of Th2 cells and cancersincluding blood cancers such as leukemia.

The present invention are described below based on Examples, but thepresent invention is not limited thereto.

Example 1 Production of Human CDR-Grafted Antibody for CCR4

1. Designing of cDNA Encoding VH and VL of Human CDR-Grafted Antibodyfor CCR4

(1) Designing of Amino Acid Sequence of VH of Human CDR-Grafted Antibodyfor CCR4

First, an amino acid sequence of the VH of a human CDR-grafted antibodyfor CCR4 (anti-CCR4 CDR-grafted antibody) was designed as follows. Anamino acid sequence of FR of VH of a human antibody was selected forgrafting amino acid sequences of CDR1, 2 and 3 of VH represented by SEQID NOs:1, 2 and 3 using the anti-CCR4 mouse antibody KM2160 (Int.Immunol., 11, 81 (1999)) established in Reference Example 1. Humanantibodies having high homology with KM2160 were retrieved from aminoacid sequence data bases of existing proteins by BLASTP method (NucleicAcid Res., 25, 3389 (1997)) using GCG Package (manufactured by GeneticsComputer Group) as a sequence analyzing system. When the homology of theactual amino acid sequence was compared with the homology scores,SWISSPROT data base accession number P01781, Ig Heavy chain V-III regionGal (Hoppe. Seylers. Z. Physiol. Chem., 354, 1505-1509 (1973);hereinafter referred to as “Gal”) was a human antibody showing thehighest homology of 82.5%, so that the FR amino acid sequence of theantibody was selected. However, positions where the amino acid residuescannot be determined uniquely (positions 28 and 30 from the N-terminalof a secretory antibody) and an amino acid residue which has lowgeneration frequency in sequences of human antibodies (Thr as the finalresidue of V region) were found in the FR amino acid sequence of Gal onthe data base. Accordingly, Ile and Ser as residues found in the mouseKM2160 were selected as positions 28 and 30, and Thr as the finalresidue of V region was substituted with Ser. Since the amino acidresidues are found at high frequencies in sequences of any humanantibodies (Sequences of Proteins of Immunological Interest, US Dep.Health and Human Services, 1991), they do not deviate from humanantibody sequences.

The VH amino acid sequence Gal0 of anti-CCR4 CDR-grafted antibodyrepresented by SEQ ID NO:4 was designed by grafting amino acid sequencesof CDR 1, 2 and 3 of VH of the anti-CCR4 mouse antibody KM2160represented by SEQ ID NOs:1, 2 and 3, respectively, to appropriatepositions in the determined human antibody FR amino acid sequence. Anucleotide sequence encoding the amino acid sequence of SEQ ID NO:4 isrepresented by SEQ ID NO:49.

Also, an amino acid sequence of VH of the anti-CCR4 CDR-grafted antibodywas designed based on the common sequences classified by Kabat et al.

Kabat et al. have classified the VH of various already known humanantibodies into three subgroups (HSG I to III) based on the homology oftheir amino acid sequences and reported common sequences in eachsubgroup (Sequences of Proteins of Immunological Interest, US Dep.Health and Human Services, 1991). There is a possibility thatimmunogenicity of the common sequences will be reduced in human.Accordingly, in order to prepare an anti-CCR4 CDR-grafted antibodyhaving high activity, among FR amino acid sequences of the commonsequences of three subgroups of the human antibody VH, an FR amino acidsequence having the highest homology with the FR amino acid sequence ofVH of KM2160 was selected in the designing. Table 1 shows a result ofthe retrieval of homology between the FR amino acid sequences of thecommon sequences of each subgroup of the human antibody VH and the FRamino acid sequence of VH of KM2160. As shown in Table 1, the FR aminoacid sequence of the VH region of KM2160 showed the highest homologywith the subgroup III.

TABLE 1 HSG I HSG II HSG III 57.47% 50.58% 77.01%

Based on the above results, the VH amino acid sequence HV0 of anti-CCR4CDR-grafted antibody represented by SEQ ID NO:38 was designed bygrafting amino acid sequence of CDR of VH of the anti-CCR4 mouseantibody KM2160 to an appropriate position of the amino acid sequence ofFR of the common sequence of subgroup III of the human antibody VH. Anucleotide sequence encoding the amino acid sequence of SEQ ID NO:38 isrepresented by SEQ ID NO:57.

(2) Designing of Amino Acid Sequence of VL of Human CDR-Grafted Antibodyfor CCR4

Next, an amino acid sequence of VL of an anti-CCR4 CDR-grafted antibodywas designed as follows. An amino acid sequence of FR of VL of a humanantibody was selected for grafting amino acid sequences of CDR1, 2 and 3of VL of anti-CCR4 mouse antibody KM2160 represented by SEQ ID NOs:5, 6and 7, respectively. Kabat et al. have classified the VL of variousalready known human antibodies into four subgroups (HSG I to IV) basedon the homology of their amino acid sequences and reported commonsequences in each subgroup (Sequences of Proteins of ImmunologicalInterest, US Dep. Health and Human Services, 1991). Accordingly, amongFR amino acid sequences of the common sequences of four subgroups of thehuman antibody VL, an FR amino acid sequence having the highest homologywith the FR amino acid sequence of VL of KM2160 was selected. Table 2shows a result of the retrieval of homology between the FR amino acidsequences of the common sequence of each subgroup of the human antibodyVL and the FR amino acid sequence of VL of KM2160. As shown in Table 2,the FR amino acid sequence of VL of KM2160 showed the highest homologywith the subgroup II.

TABLE 2 HSG I HSG II HSG III HSG IV 65.00% 82.50% 65.00% 72.50%

Based on the above results, the VL amino acid sequence LV0 of anti-CCR4CDR-grafted antibody represented by SEQ ID NO:8 was designed by graftingthe amino acid sequences of CDR1, 2 and 3 of VL of anti-CCR4 mouseantibody KM2160 represented by SEQ ID NOs: 5, 6 and 7, respectively, toappropriate positions in the amino acid sequence of FR of the commonsequence of subgroup II of the human antibody VL. A nucleotide sequenceencoding the amino acid sequence of SEQ ID NO:8 is represented by SEQ IDNO:53.

(3) Modification of VH and VL of Human CDR-Grafted Antibody for CCR4

The VH amino acid sequences Gal0 and HV0, and VL amino acid sequence LV0of anti-CCR4 CDR-grafted antibody designed in the above are antibodiesin which the CDR amino acid sequence of the anti-CCR4 mouse antibodyKM2160 alone is grafted to the selected FR amino acid sequences of humanantibody. However, when grafting with only CDR amino acid sequence of amouse antibody is carried out, the activity of a human CDR-graftedantibody is frequently decreased so that, in order to avoid thedecrease, certain amino acid residues among the FR amino acid residuesdifferent between a human antibody and a mouse antibody, which areconsidered to have influences on the activity, are generally graftedtogether with the CDR amino acid sequence. Accordingly, in this Example,an examination was carried out to identify the FR amino acid residuesconsidered to have influences on the activity.

First, three-dimensional structures of antibody V regions (Gal0LV0 andHV0LV0) comprising amino acid sequences Gal0 and HV0 of VH and aminoacid sequence LV0 of VL in the anti-CCR4 CDR-grafted antibody designedin the above were constructed using a computer modeling technique. Thethree-dimensional structure coordinates were prepared using a softwareAbM (manufactured by Oxford Molecular), and display of thethree-dimensional structures using a software Pro-Explore (manufacturedby Oxford Molecular) or RasMol (manufactured by Glaxo) according to therespective attached manufacture's instructions. Also, computer models ofthe three-dimensional structures of V regions of anti-CCR4 mouseantibody KM2160 were constructed in the same manner. In addition,three-dimensional structure models comprising modified amino acidsequences were constructed in the same manner, in which certain residuesof the FR amino acid sequences of VH and VL of Gal0LV0 or HV0LV0,different from the anti-CCR4 mouse antibody KM2160, were substitutedwith other residues found at corresponding positions in the anti-CCR4mouse antibody KM2160, and the three-dimensional structures of V regionsof the anti-CCR4 mouse antibody KM2160, Gal0LV0 or HV0LV0 and themodified product were compared.

As a result, the three-dimensional structure of the antigen bindingregion was changed so that Ala at position 40, Gly at position 42, Lysat position 43, Gly at position 44 and Lys at position 76 and Ala atposition 97 in Gal0, Thr at position 28 and Ala at position 97 for HV0and Ile at position 2, Val at position 3, Gln at position 50 and Val atposition 88 in LV0 were selected as residues considered to haveinfluence on the activity of antibody among the FR amino acid residuesof Gal0LV0 or HV0LV0. Among these selected amino acid residues, at leastone amino acid is modified into an amino acid residue(s) found in themouse antibody KM2160 so that VH and VL of human CDR-grafted antibodyhaving various modifications were designed.

First, regarding the VH, for example, Gal1 represented by SEQ ID NO:9 inwhich Ala at position 97 of Gal0 was modified, Gal2 represented by SEQID NO:10 in which Gly at position 42 and Gly at position 44 of Gal0 weremodified, Gal3 represented by SEQ ID NO:11 in which Ala at position 97,Gly at position 42 and Gly at position 44 of Gal0 were modified, HV1represented by SEQ ID NO:39 in which Thr at position 28 of HV0 wasmodified, HV2 represented by SEQ ID NO:40 in which Ala at position 97 ofHV0 was modified, and HV3 represented by SEQ ID NO:41 in which Thr atposition 28 and Ala at position 97 of HV0 were modified were designed.Furthermore, regarding the VL, for example, LV1 represented by SEQ IDNO:12 in which Ile at position 2 was modified, LV2 represented by SEQ IDNO:13 in which Val at position 3 was modified, and LV3 represented bySEQ ID NO:14 in which Ile at position 2 and Val at position 3 weremodified were designed. Nucleotide sequences encoding the amino acidsequences represented by SEQ ID NOs:9 to 11, 39 to 41 and 12 to 14 arerepresented by SEQ ID NOs:50 to 52, 58 to 60 and 54 to 56, respectively.

2, Construction of cDNA Encoding Anti-CCR4 CDR-Grafted Antibody

(1) Construction of cDNA Encoding VH of Anti-CCR4 CDR-Grafted Antibody

cDNA encoding the amino acid sequence Gal0 of VH of the anti-CCR4CDR-grafted antibody designed in 1(1) of Example 1 was constructed usingPCR as follows.

First, a complete antibody amino acid sequence by ligating the designedamino acid sequence with the H chain secretory signal sequence ofanti-CCR4 mouse antibody KM2160 represented by SEQ ID NO:15. Next, theamino acid sequence was converted into genetic codons. When two or moregenetic codons are present for one amino acid residue, a correspondinggenetic codon was determined by taking into consideration the codonusage found in nucleotide sequences of antibody genes (Sequences ofProteins of Immunological Interest, US Dep. Health and Human Services,1991). A nucleotide sequence of cDNA encoding the amino acid sequence ofcomplete antibody V region was designed by ligating the determinedgenetic codons, and binding nucleotide sequences of primers for PCRamplification (including restriction enzyme recognition sequences forcloning into a vector for humanized antibody expression) were added toits 5′-terminal and 3′-terminal. The designed nucleotide sequence wasdivided into a total of 6 nucleotide sequences each having about 100nucleotides counting from the 5′-terminal (adjoining nucleotidesequences are designed such that they have a complementary sequence ofabout 20 nucleotides on their terminal), and 6 syntheticoligonucleotides of SEQ ID NOs:16, 17, 18, 19, 20 and 21 weresynthesized in reciprocal orders of a sense chain and an antisense chain(manufactured by GENSET).

PCR was carried out by adding each oligonucleotide to a reactionsolution containing 0.2 mM dNTPs and 1 mM magnesium chloride to give afinal concentration of 0.1 μM, and adjusting the total volume to 50 μlusing 0.4 μM M13 primer RV (manufactured by Takara Shuzo), 0.4 μM M13primer M3 (manufactured by GENSET) and 2.5 units of KOD polymerase(manufactured by TOYOBO). The reaction was carried out by 30 cycles,each cycle consisting of 94° C. for 30 seconds, 55° C. for 30 secondsand 74° C. for 60 seconds, and then 1 cycle at 74° C. for 10 minutes.The reaction solution was purified using QIA quick PCR purification kit(manufactured by QIAGEN) and finally dissolved in sterile water. Thereaction solution was allowed to react at 37° C. for 1 hour using 10units of a restriction enzyme ApaI (manufactured by Takara Shuzo) and 10units of a restriction enzyme NotI (manufactured by Takara Shuzo). Thereaction solution was fractionated by agarose gel electrophoresis, andan ApaI-NotI fragment of about 0.47 kb was recovered.

Next, 3 μg of plasmid pBluescript II SK(−) (manufactured by Stratagene)was allowed to react with the fragment using 10 units of a restrictionenzyme ApaI (manufactured by Takara Shuzo) and 10 units of a restrictionenzyme NotI (manufactured by Takara Shuzo) at 37° C. for 1 hour. Saidreaction solution was fractionated by agarose gel electrophoresis, andan ApaI-NotI fragment of about 2.95 kb was recovered.

Next, the resulting ApaI-NotI fragment of the PCR product of VH of theanti-CCR4 CDR-grafted antibody and the ApaI-NotI fragment of plasmidpBluescript II SK(−) were ligated using Solution I of DNA Ligation KitVer. 2 (manufactured by Takara Shuzo) according to the manufacture'sinstructions. Escherichia coli DH5α (manufactured by TOYOBO) wastransformed using the recombinant plasmid DNA solution obtained in thismanner, each plasmid DNA was prepared from the transformant clones andthe nucleotide sequences were analyzed using Big Dye Terminator Kit ver.2 (manufactured by Applied Biosystems). As a result of the nucleotidesequence analysis, a plasmid pKM2160Gal0 shown in FIG. 1 having theobjective nucleotide sequence was obtained. Escherichia coli transformedwith pKM2160Gal0, Escherichia coli DH5α/pKM2160Gal0, has been depositedon Aug. 22, 2001, as FERM BP-7709 in International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology (AIST Tsukuba Central 6, 1-1, Higashi 1-Chome Tsukuba-shi,Ibaraki-ken 305-8566 Japan).

Next, the FR amino acid residues designed in 1(3) of Example 1 wasmodified as follows. The genetic codons for amino acid residues afterthe modification were modified to have genetic codons found in the mouseantibody KM2160.

In modification of Ala at position 97 to Gly, the PCR was carried outusing 25 ng of the plasmid pKM2160Gal0 prepared in this item as thetemplate, first heating at 94° C. for 2 minutes and then carrying out 35cycles of the reaction, each cycle consisting of 94° C. for 15 seconds,55° C. for 30 seconds and 68° C. for 40 seconds, in 50 μl of a reactionsystem prepared by adding each of the synthetic DNAs for gene transfercomprising the nucleotide sequences represented by SEQ IDs:22 and 23(manufactured by GENSET) as primers to give a final concentration of 0.4μM and using 2.5 units of KOD plus polymerase (manufactured by TOYOBO)according to the manufacture's instructions. The reaction solution waspurified using QIA quick PCR purification kit (manufactured by QIAGEN)and finally dissolved in sterile water. The total volume was allowed toreact for 1 hour at 37° C. using 10 units of a restriction enzyme PstI(manufactured by Takara Shuzo) and then allowed to react for 1 hour at37° C. using 10 units of a restriction enzyme DraIII (manufactured byNew England Biolabs). The reaction solution was fractionated by agarosegel electrophoresis, and a PstI-DraIII fragment of about 0.58 kb wasrecovered.

Next, 3 μg of the plasmid pKM2160Gal0 was allowed to react at 37° C. for1 hour using 10 units of a restriction enzyme PstI (manufactured byTakara Shuzo) and then to undergo the reaction at 37° C. for 1 hourusing 10 units of a restriction enzyme DraIII (manufactured by NewEngland Biolabs). The reaction solution was fractionated by agarose gelelectrophoresis, and a PstI-DraIII fragment of about 2.7 kb wasrecovered.

Next, the thus obtained PstI-DraIII fragment derived from the PCRproduct and the PstI-DraIII fragment derived from the plasmidpKM2160Gal0 were ligated using Solution I of DNA Ligation Kit Ver. 2(manufactured by Takara Shuzo) according to the manufacture'sinstructions. Escherichia coli DH5α (manufactured by TOYOBO) wastransformed using the recombinant plasmid DNA solution obtained in thismanner, each plasmid DNA was prepared from the transformant clones andthe nucleotide sequences were analyzed using Big Dye Terminator Kit ver.2 (manufactured by Applied Biosystems). As a result of the nucleotidesequence analysis, a plasmid pKM2160Gal1 shown in FIG. 2 having theobjective nucleotide sequence was obtained. Escherichia coli transformedwith the plasmid pKM2160Gal1, Escherichia coli DH5α/pKM2160Gal1, hasbeen deposited on Aug. 22, 2001, as FERM BP-7710 in International PatentOrganism Depositary, National Institute of Advanced Industrial Scienceand Technology (AIST Tsukuba Central 6, 1-1, Higashi 1-ChomeTsukuba-shi, Ibaraki-ken 305-8566 Japan).

In modifications of Gly at position 42 to Asp and Gly at position 44into Arg, a plasmid pKM2160Gal2 was obtained by carrying out the methodbasically similar to the above, except that the synthetic DNA for genetransfer comprising the nucleotide sequence represented by SEQ ID NO:24(manufactured by GENSET) and M13 primer RV (manufactured by TakaraShuzo) were used as the PCR primers. Escherichia coli transformed withthe plasmid pKM2160Gal2, Escherichia coli DH5α/pKM2160Gal2, has beendeposited on Aug. 22, 2001, as FERM BP-7711 in International PatentOrganism Depositary, National Institute of Advanced Industrial Scienceand Technology (AIST Tsukuba Central 6, 1-1, Higashi 1-ChomeTsukuba-shi, Ibaraki-ken 305-8566 Japan).

Also, modification of all of the above three residues was constructed asfollows. About 0.5 μg of each of the pKM2160Gal1 and pKM2160Gal2obtained in the above was allowed to react at 37° C. for 1 hour using 10units of NheI (manufactured by Takara Shuzo) and then allowed to reactat 37° C. for 1 hour using ScaI (manufactured by Takara Shuzo). Thereaction solution was fractionated by agarose gel electrophoresis, and aNheI-ScaI fragment of about 1.3 kb derived from pKM2160Gal1 and afragment of about 2.0 kb derived from pKM2160Gal2 were recovered. Theresulting two fragments were ligated using Solution I of DNA LigationKit Ver. 2 (manufactured by Takara Shuzo) according to the manufacture'sinstructions. Escherichia coli DH5α (manufactured by TOYOBO) wastransformed using the recombinant plasmid DNA solution obtained in thismanner, each plasmid DNA was prepared from the transformant clones andnucleotide sequences were analyzed using Big Dye Terminator Kit ver. 2(manufactured by Applied Biosystems). As a result of the nucleotidesequence analysis, a plasmid pKM2160Gal3 shown in FIG. 3 having theobjective nucleotide sequence was obtained. Escherichia coli transformedwith the plasmid pKM2160Gal3, Escherichia coli DH5α/pKM2160Gal3, hasbeen deposited on Aug. 22, 2001, as FERM BP-7712 in International PatentOrganism Depositary, National Institute of Advanced Industrial Scienceand Technology (AIST Tsukuba Central 6, 1-1, Higashi 1-ChomeTsukuba-shi, Ibaraki-ken 305-8566 Japan).

Next, a cDNA encoding the amino acid sequence HV0 of VH of the anti-CCR4CDR-grafted antibody designed in 1(1) of Example 1 was constructed usingPCR as follows.

First, a complete antibody amino acid sequence by ligating the designedamino acid sequence with the H chain secretory signal sequence ofanti-CCR4 mouse antibody KM2160 represented by SEQ ID NO:15. Next, theamino acid sequence was converted into genetic codons. When two or moregenetic codons are present for one amino acid residue, a correspondinggenetic codon was determined by taking into consideration the codonusage found in nucleotide sequences of antibody genes (Sequences ofProteins of Immunological Interest, US Dep. Health and Human Services,1991). The determined genetic codons were ligated so that a nucleotidesequence of cDNA encoding the amino acid sequence of complete antibody Vregion was designed, and adding nucleotide sequences of primers for PCRamplification (including restriction enzyme recognition sequences forcloning into a vector for humanized antibody expression use) were addedto its 5′-terminal and 3′-terminal. The designed nucleotide sequence wasdivided into a total of 6 nucleotide sequences each having about 100nucleotides counting from the 5′-terminal (adjoining nucleotidesequences are designed such that they have a duplication sequence ofabout 20 nucleotides on their termini) and 6 synthetic oligonucleotidesof SEQ ID NOs:16, 42, 43, 44, 45 and 21 were synthesized in reciprocalorders of a sense chain and an antisense chain (manufactured by GENSET),and then pKM2160HV0 was obtained by the method similar to pKM2160Gal0described in this item. Escherichia coli transformed with the plasmidpKM2160HV0, Escherichia coli DH5α/pKM2160HV0, has been deposited on Aug.27, 2001, as FERM BP-7718 in International Patent Organism Depositary,National Institute of Advanced Industrial Science and Technology (AISTTsukuba Central 6, 1-1, Higashi 1-Chome Tsukuba-shi, Ibaraki-ken305-8566 Japan).

In modification of Thr at position 28 to Ile, pKM2160HV1 having theobjective nucleotide sequence was obtained by carrying out the reactionsimilar to the construction of the above plasmid pKM2160HV0, using anoligonucleotide having the nucleotide sequence represented by SEQ IDNO:69 instead of the oligonucleotide having the nucleotide sequencerepresented by SEQ ID NO:42, and using an oligonucleotide having thenucleotide sequence represented by SEQ ID NO:46 instead of theoligonucleotide having the nucleotide sequence represented by SEQ IDNO:43. Escherichia coli transformed with the plasmid pKM2160HV1,Escherichia coli DH5α/pKM2160HV1, has been deposited on Aug. 27, 2001,as FERM BP-7719 in International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology (AIST TsukubaCentral 6, 1-1, Higashi 1-Chome Tsukuba-shi, Ibaraki-ken 305-8566Japan).

In modification of Thr at position 28 to Ile and Ala at position 97 toGly, pKM2160HV3 having the objective nucleotide sequence was obtained bythe reaction similar to the construction of the above plasmidpKM2160HV0, using an oligonucleotide having the nucleotide sequencerepresented by SEQ ID NO:69 instead of the oligonucleotide having thenucleotide sequence represented by SEQ ID NO:42, using anoligonucleotide having the nucleotide sequence represented by SEQ IDNO:46 instead of the oligonucleotide having the nucleotide sequencerepresented by SEQ ID NO:43 and using an oligonucleotide having thenucleotide sequence represented by SEQ ID NO:47 instead of theoligonucleotide having the nucleotide sequence represented by SEQ IDNO:45. Escherichia coli transformed with the plasmid pKM12160HV3,Escherichia coli DH5α/pKM2160HV3, has been deposited on Aug. 27, 2001,as FERM BP-7721 in International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology (AIST TsukubaCentral 6, 1-1, Higashi 1-Chome Tsukuba-shi, Ibaraki-ken 305-8566Japan).

In modification of Ala at position 97 to Gly, the plasmid wasconstructed as follows. About 0.5 μg of each of the obtained pKM2160HV0and pKM2160HV3 was allowed to react at 37° C. for 1 hour using 10 unitsof a restriction enzyme NheI (manufactured by Takara Shuzo) and thenallowed to react at 37° C. for 1 hour using ScaI (manufactured by TakaraShuzo). The reaction solution was fractionated by agarose gelelectrophoresis, and a NheI-ScaI fragment of about 1.3 kb derived frompKM2160HV3 and a fragment of about 2.0 kb derived from pKM2160HV0 wererecovered. The resulting two fragments were ligated using Solution I ofDNA Ligation Kit Ver. 2 (manufactured by Takara Shuzo) according to themanufacture's instructions. Escherichia coli DH5α (manufactured byTOYOBO) was transformed using the recombinant plasmid DNA solutionobtained in this manner, each plasmid DNA was prepared from thetransformant clones and nucleotide sequences were analyzed using Big DyeTerminator Kit ver. 2 (manufactured by Applied Biosystems). As a resultof the nucleotide sequence analysis, a plasmid pKM2160HV2 having theobjective nucleotide sequence was obtained. Escherichia coli transformedwith the plasmid pKM2160HV2, Escherichia coli DH5α/pKM2160HV2, has beendeposited on Aug. 27, 2001, as PERM BP-7720 in International PatentOrganism Depositary, National Institute of Advanced Industrial Scienceand Technology (AIST Tsukuba Central 6, 1-1, Higashi 1-ChomeTsukuba-shi, Ibaraki-ken 305-8566 Japan).

(2) Construction of cDNA Encoding VL of Anti-CCR4 CDR-Grafted Antibody

A cDNA encoding the amino acid sequence LV0 of VL of the anti-CCR4CDR-grafted antibody designed in 1(2) of Example 1 was constructed usingPCR similar to the case of VH as follows. In this case, the L chainsequence of anti-CCR4 mouse antibody KM2160 having the amino acidsequence represented by SEQ ID NO:25 was used as the secretory signalsequence.

First, 6 synthetic oligonucleotides having the nucleotide sequencesdescribed in SEQ ID NOs:26, 27, 28, 29, 30 and 31 were synthesized(manufactured by GENSET). PCR was carried out by adding eacholigonucleotide to 50 μl of a reaction solution to give a finalconcentration of 0.1 μM, and using 0.4 μM of M13 primer RV (manufacturedby Takara Shuzo) and 0.4 μM of M13 primer M4 (manufactured by TakaraShuzo) or M13 primer M3 (manufactured by GENSET) represented by SEQ IDNO:32 and 2.5 units of KOD polymerase (manufactured by TOYOBO). Thereaction was carried out by 30 cycles, each cycle consisting of 94° C.for 30 seconds, 55° C. for 30 seconds and 74° C. for 60 seconds, andsubsequent 1 cycle at 72° C. for 10 minutes. The reaction solution waspurified using QIA quick PCR purification kit (manufactured by QIAGEN)and finally dissolved in sterile water. The reaction solution wasallowed to react using 10 units of a restriction enzyme EcoRI(manufactured by Takara Shuzo) and 10 units of a restriction enzyme XhoI(manufactured by Takara Shuzo) at 37° C. for 1 hour. The reactionsolution was fractionated by agarose gel electrophoresis, and anEcoRI-XhoI fragment of about 0.44 kb was recovered.

Next, 3 μg of the plasmid pBluescript II SK(−) (manufactured byStratagene) was allowed to react using 15 units of a restriction enzymeEcoRI (manufactured by Takara Shuzo) and 15 units of a restrictionenzyme XhoI (manufactured by Takara Shuzo) at 37° C. for 1 hour. Thereaction solution was fractionated by agarose gel electrophoresis, andan EcoRI XhoI fragment of about 2.95 kb was recovered.

Next, the resulting EcoRI-XhoI fragment of the PCR product of VL of theanti-CCR4 CDR-grafted antibody and the EcoRI-XhoI fragment of plasmidpBluescript II SK(−) were ligated using Solution I of DNA Ligation KitVer. 2 (manufactured by Takara Shuzo) according to the manufacture'sinstructions. Escherichia coli DH5α (manufactured by TOYOBO) wastransformed using the recombinant plasmid DNA solution obtained in thismanner, each plasmid DNA was prepared from the transformant clones andnucleotide sequences were analyzed using Big Dye Terminator Kit ver. 2(manufactured by Applied Biosystems). As a result of the nucleotidesequence analysis, a plasmid pKM2160LV4 shown in FIG. 4 having theobjective nucleotide sequence was obtained. Escherichia coli transformedwith pKM2160LV0, Escherichia coli DH5α/pKM2160LV0, has been deposited onAug. 22, 2001, as FERM BP-7713 in International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology (AIST Tsukuba Central 6, 1-1, Higashi 1-Chome Tsukuba-shi,Ibaraki-ken 305-8566 Japan).

Next, the FR amino acid residues designed in 1(3) of Example 1 weremodified as follows. The genetic codons for amino acid residues afterthe modification were modified to have genetic codons found in the mouseantibody KM2160.

In modification of IIe at position 2 to Val, a plasmid pKM2160LV1 shownin FIG. 5 having the objective nucleotide sequence was obtained bycarrying out the reaction similar to the construction of the aboveplasmid pKM2160LV0, using an oligonucleotide having the nucleotidesequence represented by SEQ ID NO:33 instead of the oligonucleotidehaving the nucleotide sequence represented by SEQ ID NO:27. Escherichiacoli transformed with the plasmid pKM2160LV1, Escherichia coliDH5α/pKM2160LV1, has been deposited on Aug. 22, 2001, as FERM BP-7714 inInternational Patent Organism Depositary, National Institute of AdvancedIndustrial Science and Technology (AIST Tsukuba Central 6, 1-1, Higashi1-Chome Tsukuba-shi, Ibaraki-ken 305-8566 Japan).

In the same manner, each of the objective plasmids pKM2160LV2 andpKM2160LV3 was obtained by using an oligonucleotide having thenucleotide sequence represented by SEQ ID NO:34 instead of theoligonucleotide having the nucleotide sequence represented by SEQ IDNO:27 when Val at position 3 was modified into Leu, and using anoligonucleotide having the nucleotide sequence represented by SEQ IDNO:35 instead of the oligonucleotide having the nucleotide sequencerepresented by SEQ ID NO:27 when both of the above two residues weremodified. Escherichia coli transformed with the plasmid pKM2160LV2,Escherichia coli DH5α/pKM2160LV2, and Escherichia coli transformed withthe plasmid pKM2160LV3, Escherichia coli DH5α/pKM2160LV3, have beendeposited on Aug. 22, 2001, as FERM BP-7715 and FERM BP-7716,respectively, in International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology (AIST TsukubaCentral 6, 1-1, Higashi 1-Chome Tsukuba-shi, Ibaraki-ken 305-8566Japan).

(3) Construction of Anti-CCR4 CDR-Grafted Antibody Expression Vector

An anti-CCR4 CDR-grafted antibody expression vector pKANTEX2160Gal0LV0was constructed using a vector for humanized antibody expressionpKANTEX93 (Mol. Immunol., 37, 1035 (2000)) and the plasmids pKM2160Gal0and pKM2160LV0 obtained in 2(1) and (2) of Example 1 as follows.

The plasmid pKM2160LV0 (3 μg) obtained in 2(2) of Example 1 was allowedto react with 10 units of a restriction enzyme BsiWI (manufactured byNew England Biolabs) at 55° C. for 1 hour and then with 10 units of arestriction enzyme EcoRI (manufactured by Takara Shuzo) at 37° C. for 1hour. The reaction solution was fractionated by agarose gelelectrophoresis, and a BsiWI-EcoRI fragment of about 0.44 kb wasrecovered.

Next, 3 μg of the vector for humanized antibody expression pKANTEX93 wasallowed to react with 10 units of a restriction enzyme BsiWI(manufactured by New England Biolabs) at 55° C. for 1 hour and then with10 units of a restriction enzyme EcoRI (manufactured by Takara Shuzo) at37° C. for 1 hour. The reaction solution was fractionated by agarose gelelectrophoresis, and a BsiWI-EcoRI fragment of about 12.75 kb wasrecovered.

Next, the resulting BsiWI-EcoRI fragment derived from pKM2160LV0 and theBsiWI-EcoRI fragment derived from pKANTEX93 were ligated using Solution1 of DNA Ligation Kit Ver. 2 (manufactured by Takara Shuzo) according tothe manufacture's instructions. Escherichia coli DH5α (manufactured byTOYOBO) was transformed using the recombinant plasmid DNA solutionobtained in this manner to thereby obtain a plasmid pKANTEX2160LV0 shownin FIG. 6.

Next, 3 μg of the plasmid pKM2160Gal0 obtained in 2(1) of Example 1 wasallowed to react with 10 units of a restriction enzyme ApaI(manufactured by Takara Shuzo) at 37° C. for 1 hour and then with 10units of a restriction enzyme NotI (manufactured by Takara Shuzo) at 37°C. for 1 hour. The reaction solution was fractionated by agarose gelelectrophoresis, and an ApaI-NotI fragment of about 0.47 kb wasrecovered.

Next, 3 μg of the plasmid pKANTEX2160LV0 obtained in the above wasallowed to react with 10 units of a restriction enzyme ApoI(manufactured by Takara Shuzo) at 37° C. for 1 hour and then with 10units of a restriction enzyme NotI (manufactured by Takara Shuzo) at 37°C. for 1 hour. The reaction solution was fractionated by agarose gelelectrophoresis, and an ApaI-NotI fragment of about 0.45 kb wasrecovered.

Next, the resulting ApaI-NotI fragment derived from the pKM2160Gal0 andthe ApaI-NotI fragment derived from the plasmid pKANTEX2160LV0 wereligated using Solution I of DNA Ligation Kit Ver. 2 (manufactured byTakara Shuzo) according to the manufacture's instructions. Escherichiacoli DH5α (manufactured by TOYOBO) was transformed using the recombinantplasmid DNA solution obtained in this manner, and each plasmid DNA wasprepared from the transformant clones.

As a result that the nucleotide sequences of the thus obtained plasmidswere analyzed using Big Dye Terminator Kit ver. 2 (manufactured byApplied Biosystems), it was confirmed that an expression vectorpKANTEX2160Gal0LV0 shown in FIG. 6 into which the objective DNA had beencloned was obtained.

In addition, expression vectors were prepared using the same method onthe VH and VL in which amino acid residues of other FR were modified,including HV0.

Specifically, 22 expression vectors pKM2160Gal0LV0, pKM2160Gal0LV1,pKM2160Gal0LV2, pKM2160Gal0LV3, pKM2160Gal1LV1, pKM2160Gal1LV3,pKM2160Gal2LV1, pKM2160Gal2LV3, pKM2160Gal3LV1, pKM2160Gal3LV3,pKM2160HV0LV0, pKM12160HV0L V 1, pKM2160HV0LV2, pKM2160HV0LV3,pKM2160HV1LV0, pKM2160HV1LV1, pKM2160HV1LV2, pKM2160HV1LV3,pKM2160HV2LV0, pKM2160HV2LV3, pKM2160HV3LV0 and pKM2160HV3LV3 wereconstructed by respectively combining pKM2160Gal0, pKM2160Gal1,pKM2160Gal2, pKM2160Gal3, pKM2160HV0, pKM2160HV1, pKM2160HV2 andpKM2160HV3 constructed in 2(1) of Example 1 with the pKM2160LV0,pKM2160LV1, pKM2160LV2 and pKM2160LV3 constructed in 2(2) of Example 1.

Example 2 Expression of Anti-CCR4 CDR-Grafted Antibody in Animal Cells

1. Transient Expression of Anti-CCR4 CDR-Grafted Antibody Using COS-7Cell (ATCC CRL 1651)

(1) Transient Expression at COS-7 Cell

Into a 6 well plate (manufactured by Iwaki Glass), 1×10⁵ cells/ml ofCOS-7 cell was dispensed at 2 ml/well using DMEM medium (manufactured byBibco) containing 10% FCS and cultured overnight at 37° C. Per 100 μl ofOPTI-MEM medium (manufactured by Bibco), 3 μl of Fu-GENE™ 6 TransferReagent (manufactured by Roche) was added and 1 μg of each of the 22anti-CCR4 CDR-grafted antibody expression vectors obtained in thearticle 2(3) of Example 1 was further added thereto, and the mixture wasallowed to stand at room temperature for 15 minutes to form aDNA-liposome complex. Each of the reaction solutions was added dropwiseto the above COS-7 cell and thoroughly mixed, followed by culturing at37° C. After the culturing for 72 hours, the culture supernatants wererecovered and the activity of the anti-CCR4 CDR-grafted antibodyactivity in the culture supernatants was evaluated.

(2) Reactivity Evaluation of Anti-CCR4 CDR-Grafted Antibody for HumanCCR4

The activity of the resulting culture supernatants of 22 antibodies wasevaluated as follows.

Compound 1 (SEQ ID NO:37) was selected as a human CCR4 extracellularregion peptide which can react with the anti-CCR4 chimeric antibodyKM2760 produced by the transformant KM2760 (FERM BP-7054) prepared inReference Example 2. In order to use Compound 1 in the activitymeasurement by ELISA, its conjugate with BSA (bovine serum albumin)(manufactured by Nakalai Tesque) was prepared and used as the antigen.That is, 100 ml of 25 mg/ml SMCC(4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid N-hydroxysuccinimideester) (manufactured by Sigma)-DMSO solution was added dropwise to 900ml of PBS solution containing 10 mg BSA under vortex and gently stirredfor 30 minutes. To a gel filtration column, such as NAP-10 column or thelike, equilibrated with 25 ml of PBS, 1 ml of the reaction solution wasapplied, and the eluate eluted with 1.5 ml of PBS was used as a BSA-SMCCsolution (BSA concentration was calculated by A₂₈₀ measurement). Next,250 ml of PBS was added to 0.5 mg of Compound 1 which was thencompletely dissolved by adding 250 ml of DMF, and then the aboveBSA-SMCC solution (BSA content: 1.25 mg) was added under vortex andgently stirred for 3 hours. The reaction solution was dialyzed overnightat 4° C. against PBS, sodium azide was added thereto to give a finalconcentration of 0.05% and then the resulting mixture was filtered usinga 0.22 mm filter to give a BSA-Compound 1 solution.

Into a 96 well ELISA plate (manufactured by Greiner), 0.05 μg/ml of theprepared conjugate was dispensed in at 50 μl/well and allowed to standat 4° C. overnight for adsorption. After washing with PBS, PBScontaining 1% BSA (hereinafter referred to as “1% BSA-PBS”) was addedthereto at 100 μl/well and allowed to react at room temperature for 1hour to block the remaining active groups. After washing each well withPBS containing 0.05% Tween 20 (hereinafter referred to as “Tween-PBS”),a culture supernatant of the transformant was added at 50 μl/well andallowed to react at room temperature for 1 hour. After the reaction andsubsequent washing of each well with Tween-PBS, a peroxidase-labeledgoat anti-human IgG(γ) antibody solution (manufactured by AmericanQualex) diluted 6,000 folds with 1% BSA-PBS was added as a secondaryantibody solution in 50 μl/well portions and allowed to react at roomtemperature for 1 hour. After the reaction and subsequent washing withTween-PBS, an ABTS substrate solution (a solution prepared by dissolving0.55 g of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)ammonium in 1 liter of 0.1 M citrate buffer (pH 4.2) and adding 1 μl/mlhydrogen peroxide just before use) was added at 50 μl/well to developcolor, and the reaction was stopped 20 minutes thereafter by adding 5%SDS solution at 50 μl/well. Thereafter, absorbance at 415 nm wasmeasured.

Also, in order to compare concentrations of a produced human IgGantibody in the culture supernatants, a goat anti-human IgG(γ) antibody(manufactured by American Qualex) diluted 2,000 folds with PBS was usedas the antigen.

The results are shown in FIG. 7 and FIG. 8. Each human CCR4 CDR-graftedantibody showed almost the same activity of the human chimeric antibodyKM2760.

2. Stable Expression of Anti-CCR4 CDR-Grafted Antibody Using AnimalCells

An anti-CCR4 CDR-grafted antibody was expressed in animal cells usingthe anti-CCR4 CDR-grafted antibody expression vector obtained in 2(3) ofExample 1 as follows.

(1) Stable Expression in Rat Myeloma Cell Line YB2/0 Cell (ATCC CRL1581)

Each human CDR-grafted antibody expression plasmid was made into alinear state by digesting it with a restriction enzyme AatII(manufactured by TOYOBO), 10 μg of the digested product was introducedinto 4×10⁶ cells of the rat myeloma cell line YB2/0 cell (ATCC CRL 1581)by electroporation (Cytotechnology, 3, 133 (1990)), and then the cellswere suspended in 40 ml of H-SFM (manufactured by GIBCO-BRL) medium(supplemented with 5% of fetal bovine serum (FBS)) and dispensed at 200μl/well into a 96 well microtiter plate (manufactured by SumitomoBakelite). After culturing at 37° C. for 1 to 3 days in a 5% CO₂incubator, G418 (manufactured by Nakalai Tesque) was added thereto togive a concentration of 1 mg/ml and the culturing was continued for 1 to2 weeks to obtain G418-resistant transformants.

Culture supernatants were recovered from wells in which colonies of thetransformants showing G418 resistance became confluent, andantigen-binding activities of anti-CCR4 human CDR-grafted antibodies inthe culture supernatants were measured by the ELISA shown in 1(2) ofExample 2.

In order to increase the antibody expression amount using a dhfr geneamplification system, the transformants in wells in which expression ofan anti-CCR4 chimeric antibody was found in the culture supernatants wassuspended to give a density of 1 to 2×10⁵ cells/ml in H-SFM mediumcontaining 1 mg/ml G418 and 50 nM methotrexate (hereinafter referred toas “MTX”) which is an inhibitor of a dhfr gene product dihydrofolatereductase and dispensed in 1 ml portions into a 24 well plate(manufactured by Greiner). Transformants showing 50 nM MTX resistancewere induced by culturing at 37° C. for 1 to 2 weeks in a 5% CO₂incubator. When the transformants became confluent in the wells,antigen-binding activities of anti-CCR4 human CDR-grafted antibodies inthe culture supernatants were measured by the ELISA shown in 1(2) ofExample 2. Regarding transformants of wells where expression ofanti-CCR4 human CDR-grafted antibodies was found in the culturesupernatants, the MTX concentration was increased to 100 nM and then to200 nM by the above method, and a transformant capable of growing inH-SFM medium containing 1 mg/ml G418 and 200 nM MTX and also capable ofhighly expressing the anti-CCR4 human CDR-grafted antibody was finallyobtained. For the thus obtained transformant, single cell isolation(cloning) was carried out by limiting dilution analysis to obtain atransformant cell clone showing the highest expression of the anti-CCR4human CDR-grafted antibody. An antibody producing cell KM8760 obtainedby the gene transfer of an expression vector pKANTEX2160Gal1LV3 and anantibody producing cell KM8759 obtained by the gene transfer of anexpression vector pKANTEX2160Gal2LV3 have been deposited on Jul. 30,2002, as FERM BP-8130 and FERM BP-8129, respectively, in theInternational Depositary Authority at International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology (AIST Tsukuba Central 6, 1-1, Higashi 1-Chome Tsukuba-shi,Ibaraki-ken 305-8566 Japan).

(2) Purification of Anti-CCR4 CDR-Grafted Antibody from CultureSupernatant

When a transformant showing G418 resistance appeared and becameconfluent, the medium was changed to 300 to 1,100 ml of H-SFM mediumcontaining Daigo's GF21 (manufactured by Wako Pure Chemical Industries)at a concentration of 5%, followed by culturing for 3 to 5 days. When itbecame confluent, the culture supernatant was recovered. A purifiedprotein was obtained by purifying the anti-CCR4 CDR-grafted antibodyfrom about 300 to 1,100 ml of the culture supernatant using Prosep-Acolumn (manufactured by Millipore) in accordance with the attachedinstructions.

3. Activity Evaluation of Purified Anti-CCR4 CDR-Grafted Antibody

The activity was evaluated using anti-CCR4 CDR-grafted antibodiesderived from antibody-producing cells obtained by introducing expressionvectors of Gal0LV0, Gal0LV1, Gal0LV3, Gal1LV1, Gal1LV3, Gal2LV1,Gal2LV3, Gal3LV1 and Gal3LV3 into YB2/0 (hereinafter referred simply toas “Gal0LV0”, “Gal0LV1”, “Gal0LV3”, “Gal1LV1”, “Gal2LV1”, “Gal2LV3”,“Gal3LV1” and “Gal3LV3”, respectively).

(1) Measurement of Binding Activity of Anti-CCR4 CDR-Grafted Antibodyfor Human CCR4 (ELISA Method)

The measurement was carried out in the same manner as the methoddescribed in the article 1(2) of Example 2. The results are shown inFIG. 9. Each anti-CCR4 CDR-grafted antibody showed almost the sameactivity of that of the human chimeric antibody KM2760.

(2) Reactivity of Anti-CCR4 CDR-Grafted Antibody with HumanCCR4-High-Expressing Cell (Fluorescent Antibody Technique)

Into a 96 well plate, 2×10⁵ or more of CCR4/EL-4 cells,CCR4-high-expressing cells obtained in Reference Example 3, weredispensed. An antibody solution was prepared by diluting each of thepurified antibodies and a human immunoglobulin (manufactured by Welfide)for preventing nonspecific staining to give concentrations of 10 μg/mland 3.75 mg/ml, respectively, with a buffer for FACS, and the antibodysolution was added at 100 μl/well and allowed to react for 30 minutes inice. As a negative control, 10 μg/ml of an anti-human IL-5 receptor ofchain antibody (WO 97/10354) was used. After washing twice with 200μl/well of the buffer for FACS, a PE-labeled anti-human IgG antibody(manufactured by Coulter) diluted 100 times was added at 50 μl/well.After the reaction in ice under shading and subsequent washing threetimes with 200 μl/well of the buffer for FACS, the reaction product wassuspended in 500 μl of the buffer for FACS and the fluorescenceintensity was measured using a flow cytometer. The results are shown inFIG. 10. All of the anti-CCR4 CDR-grafted antibodies showed almost thesame activity as the human chimeric antibody KM2760.

(3) Measurement of Activity of Anti-CCR4 CDR-Grafted Antibody to Bind toHuman CCR4 (BIAcore Method)

In order to measure the binding activity in more detail, the bindingactivity of various purified antibodies was measured using BIAcore 2000(manufactured by BIACORE) as follows. In this case, HBS-EP (manufacturedby BIACORE) was used as the buffer for diluting samples and for themeasurement. First, 5 μl of 0.05 μg/ml solution of Compound 1 as abiotinylated CCR4 partial peptide was added to a sensor tip SA(manufactured by BIACORE) at a flow rate of 5 μl/minute and immobilizedon the sensor tip.

To the prepared biotinylated compound 1-immobilized sensor tip, 20 μl of4 μg/ml solution of each of the purified antibodies was added at a flowrate of 5 μl/minute, and after completion of the addition, thedissociation reaction was monitored for 4 minutes and then the sensortip surface was regenerated by adding 5 μl of 10 mM HCl twice. In thisway, a binding reaction curve (sensorgram) for Compound 1 was obtained.

The results are shown in FIG. 11. The ordinate represents a resonanceunit (RU) which means a mass change on the sensor tip. For example,1,000 RU corresponds to a mass change of about 1 ng/mm² protein. It wasshown that KM 2760 has a markedly stable and high binding activity,because it showed a time-dependent binding activity for Compound 1 anddissociation of its binding was hardly found by the dissociationreaction. On the other hand, each of the anti-CCR4 CDR-graftedantibodies showed almost the same dissociation reaction as the humanchimeric antibody KM2760, but a slight decrease in the activity wasfound in the binding reaction to the CCR4 partial peptide, Compound 1.The anti-CCR4 CDR-grafted antibody Gal0LV0 in which CDR alone wasgrafted showed the lowest binding activity, and the binding activity wasincreased by the amino acid residue modification of FR. The results showthat an anti-CCR4 CDR-grafted antibody which maintains the antigenbinding activity and binding specificity of a mouse antibody can beprepared by grafting CDR of the mouse antibody KM2160 to an appropriatehuman antibody FR, and that an anti-CCR4 CDR-grafted antibody havinghigher binding activity can be prepared by identifying FR amino acidresidues important for the binding activity based on thethree-dimensional structure and the like of antibody V regions andgrafting them together with the CDR. It is expected that the anti-CCR4CDR-grafted antibodies prepared in this Example have high bindingactivity to CCR4, have decreased immunogenicity in human in comparisonwith that of mouse antibodies and also that human chimeric antibodies,and have high safety and high therapeutic effects.

2. In Vitro Cytotoxic Activity of Anti-CCR4 CDR-Grafted Antibody (ADCCActivity)

In order to evaluate in vitro cytotoxic activity of the purifiedanti-CCR4 CDR-grafted antibody obtained in 2(2) of Example 2, its ADCCactivity was measured as follows.

(1) Preparation of Target Cell Suspension

The human CCR4-high-expressing cell CCR4/EL-4 obtained in ReferenceExample 3 was cultured in 10% FCS-containing RPMI 1640 medium(manufactured by GIBCO) containing 0.5 mg/ml G418 to give a density of1×10⁶ cells/0.5 ml, 1.85 MBq equivalent of radioactive sodium chromate(Na₂ ⁵¹CrO₄) (manufactured by Daiichi Pure Chemicals) was added theretoand the mixture was allowed to react at 37° C. for 1.5 hours toisotope-label the cells. After the reaction, the cells were washed threetimes by their suspension in RPMI 1640 medium and centrifugation,re-suspended in the medium and then incubated in ice at 4° C. for 30minutes to spontaneously release the radioactive substance. Aftercentrifugation, S ml of 10% FCS-containing RPMI 1640 medium was addedthereto to give a density of 2×10⁵ cells/ml as the target cellsuspension.

(2) Preparation of Effector Cell Suspension

Healthy human peripheral blood (60 ml) was collected using a syringecontaining 200 units (200 μl) of a heparin sodium injection(manufactured by Takeda Pharmaceutical). The entire amount was filled upto 120 ml by diluting it two folds with the same volume of physiologicalsaline (manufactured by Otsuka Pharmaceutical). Lymphoprep (manufacturedby NYCOMED) was dispensed at 5 ml into 12 tubes of 15 ml capacitycentrifugation tubes (manufactured by Sumitomo Bakelite), the dilutedperipheral blood was over-layered thereon at 10 ml, and the mixture wascentrifuged at 800×g for 20 minutes at room temperature. PBMC fractionsbetween the blood plasma layer and the Lymphoprep layer were collectedfrom all centrifugation tubes, suspended in 1% FCS-containing RPMI 1640medium (hereinafter referred to as “1% FCS-RPMI”), washed twice bycentrifugation at 400×g and 4° C. for 5 minutes and then re-suspended togive a density of 5×10⁶ cells/ml to be used as the effector cells.

(3) Measurement of ADCC Activity

The target cell suspension prepared in (1) was dispensed at 50 μl (1×10⁴cells/well) into wells of a 96 well U bottom plate (manufactured byFalcon). Next, the effector cell suspension prepared in (2) wasdispensed at 100 μl (5×10⁵ cells/well, the ratio of effector cells totarget cells becomes 50:1). Next, each anti-CCR4 chimeric antibody wasadded to give a final concentration of 0.1 ng/ml to 10 μg/ml and themixture was allowed to react at 37° C. for 4 hours. After the reaction,the plate was centrifuged and the amount of ⁵¹Cr in 100 μl of thesupernatant in each well was measured by a γ-counter. The amount of thespontaneously dissociated ⁵¹Cr was calculated in the same manner as theabove using the medium alone instead of the effector cell suspension andantibody solution and measuring the amount of ⁵¹Cr in the supernatant.The amount of the total dissociated ⁵¹Cr was calculated in the samemanner as the above by adding the medium alone instead of the antibodysolution, and 1 N hydrochloric acid instead of the effector cellsuspension, and measuring the amount of ⁵¹Cr in the supernatant. TheADCC activity was calculated by the following equation:

${{ADCC}\mspace{14mu}{{activity}(\%)}} = \frac{\begin{matrix}{\left( {{amount}\mspace{14mu}{of}\mspace{14mu}{\,^{51}{Cr}}\mspace{14mu}{in}\mspace{14mu}{sample}{\mspace{11mu}\;}{supernatant}} \right) -} \\\left( {{amount}\mspace{14mu}{of}\mspace{14mu}{spontaneously}\mspace{14mu}{released}\mspace{14mu}{\,^{51}{Cr}}} \right)\end{matrix}}{\begin{matrix}{\left( {{amount}\mspace{14mu}{of}\mspace{14mu}{total}\mspace{14mu}{\,^{51}{Cr}}} \right) -} \\\left( {{amount}\mspace{14mu}{of}\mspace{14mu}{spontaneously}\mspace{14mu}{released}\mspace{14mu}{\,^{51}{Cr}}} \right)\end{matrix}}$

The results are shown in FIG. 12. As shown in FIG. 12, the anti-CCR4CDR-grafted antibody had strong cytotoxic activityantibody-concentration-dependently.

5. Effect of Inhibiting Production of Cytokine from Human PBMC

An effect of inhibiting cytokine production was examined using ananti-CCR4 CDR-grafted antibody Gal1LV3 and a chimeric antibody KM2760.An anti-IL-5R antibody was used as a negative control.

PBMC was separated in the same manner as in 4(2) of Example 2 anddispensed at 1×10⁶ cells/well into a 96 well U-bottom plate, anevaluation antibody was added thereto to give a final concentration of 1μg/ml, and the total volume was adjusted to 200 μl/well. The ADCCactivity was induced by co-culturing at 37° C. for 24 hours in a streamof 5% CO₂. After the culturing, 100 μl of the supernatant was removed,100 μl of a medium containing 100 ng/ml PMA (phorbol myristate acetate)and 2 μg/ml ionomycin (manufactured by SIGMA) was added thereto to givefinal concentrations of 50 ng/ml PMA and 1 μg/ml ionomycin, and thecells were stimulated to induce the cytokine production. Afterintroduction of each stimulant, culturing was carried out for 24 hours,culture supernatants were recovered and IL-4, IL-13 and interferon(IFN)-γ were measured using a cytokine assay kit (manufactured byBiosource). The production inhibition ratio was calculated by definingeach cytokine production in the absence of antibody as 0% inhibitionratio, and the results are shown in FIG. 13. As shown in FIG. 13,similar to the chimeric antibody KM2760, the anti-CCR4 CDR-graftedantibody Gal1LV3-added group significantly inhibited production of theTh2 cytokine IL-4 and IL-13 but had little influence on the Th1 cytokineIFN-γ.

The results show that each anti-CCR4 CDR-grafted antibody can deplete oreliminate CCR4-expressed Th2 cells by activating human effector cellsefficiently and, as a result, has an effect of inhibiting production ofTh2 cytokine from the Th2 cells and therefore is useful in diagnosing ortreating human Th2-mediated immune diseases such as bronchial asthma,atopic dermatitis and the like.

6. Analysis of Reactivity for Human Platelet

(1) Separation of Human Platelet

A 1/10 volume of 3.2% sodium citrate was added to a blood samplecollected from a healthy parson and thoroughly mixed. The blood wasdispensed at 5 ml into 15 ml capacity tubes (manufactured by Greiner)and centrifuged at 90×g for 10 minutes at room temperature. Thesupernatants were collected and further centrifuged at 1,950×g for 10minutes at room temperature. After discarding the supernatant, thepellets were suspended in the buffer for FACS and centrifuged at 1,190×gfor 5 minutes at room temperature to wash the pellets. The pellets wereagain suspended in the buffer for FACS and centrifuged in the samemanner, and then platelets in the pellet form were adjusted to give adensity of about 1×10⁷ pellets/ml using the buffer for FACS.

(2) Staining of Platelet

Each of the purified anti-CCR4 human CDR-grafted antibodies obtained in3 in Example 2 was added to 100 μl of the platelet suspension obtainedin the item 6(1) to give a concentration of 10 μg/100 μl and allowed toreact at room temperature for 30 minutes in the dark. As comparativecontrols, each of an anti-CCR4 human chimeric antibody KM2760 and ananti-mouse antibody IG1 antibody (manufactured by Pharmingen) wasallowed to react with 100 ml of the platelet suspension at the sameconcentration. After the reaction, 2 ml of the buffer for FACS was addedto each of 15 ml capacity tubes, and the mixture was stirred and thenwashed by centrifugation at 840×g for 5 minutes at 4° C. Afterdiscarding the supernatant, the same operation was carried out again. APE-labeled anti-mouse IgG antibody (manufactured by Coulter) diluted 50folds with the buffer for FACS was added at 20 μl the tube containing asample reacted with each of the human CDR-grafted antibodies and KM2760and allowed to react at room temperature for 30 minutes in the dark.Regarding the tube containing the sample reacted with the 1G1 antibody,20 μl of a 50 times-diluted PE-labeled anti-mouse IgG antibody(manufactured by DAKO) was further added and allowed to react at roomtemperature for 30 minutes in the dark.

After the reaction, 2 ml of the buffer for FACS was added to each tube,followed by stirring, and the mixture was washed by centrifugation at840×g for 5 minutes at 4° C. After discarding the supernatant, the sameoperation was carried out again. After suspending the residue in 500 μlof the buffer for FACS, the fluorescence intensity was measured using aflow cytometer EPICS XL-MCL (manufactured by Beckman Coulter).

The results are shown in FIG. 14. The 1G1 antibody as a comparativecontrol showed reactivity with platelets, but all of the anti-CCR4 humanCDR-grafted antibodies did not show specific reactivity with humanplatelets similar to the anti-CCR4 human chimeric antibody KM2760.

Reference Example 1

Preparation of Hybridoma Cell which Produces Mouse Anti-CCR4 MonoclonalAntibody

Hybridoma cells which produce mouse anti-CCR4 monoclonal antibody KM2160(Int. Immunol., 11, 81 (1999)) were produced according to the followingprocedure.

(1) Preparation of Antigen

The amino acid sequence (SEQ ID NO:48) of human CCR4 (hereinafterreferred to as “hCCR4”) protein was analyzed by using Genetyx Mac, andCompound 2 (SEQ ID NO:36) was selected as a partial sequence consideredto be appropriate as the antigen among parts having high hydrophilicity,N-terminal and C-terminal.

(2) Preparation of Immunogen

The hCCR4 partial peptide obtained in (1) of Reference Example 1 wasused as the immunogen after preparing its conjugate with KLH(Calbiochem) by the following method in order to increase itsimmunogenicity. Specifically, KLH was dissolved in PBS to give aconcentration of 10 mg/ml, 1/10 volume of 25 mg/ml MBS (manufactured byNakalai Tesque) was added dropwise thereto, and the mixture was allowedto react by stirring for 30 minutes. Free MBS was removed by a gelfiltration column such as Sephadex G-25 column which had beenequilibrated in advance with PBS or the like, and 2.5 mg of theresulting KLH-MB was mixed with 1 mg of the peptide dissolved in 0.1 Msodium phosphate buffer (pH 7.0), followed by stirring at roomtemperature for 3 hours. After the reaction, the mixture was dialyzedagainst PBS.

(3) Immunization of Animal and Production of Antibody-Producing Cells

To 5-weeks-old female mice (Balb/c), 100 μg of the peptide-KLH conjugateprepared in (2) of Reference Example 1 was administered together with 2mg of aluminum gel and 1×10⁹ cells of pertussis vaccine (manufactured byChiba Serum Institute), and 2 weeks thereafter, 100 μg of the conjugatewas administered once a week at a total of 4 times. A blood sample wastaken from each animal from the venous plexus of the fundus of the eye,its serum titer was examined by an enzyme immunoassay described below,and the spleen was excised 3 days after the final immunization from amouse which showed a sufficient antibody titer. The spleen was excisedfrom a mouse on the 3rd day after the final administration and cut topieces in MEM (manufactured by Nissui Pharmaceutical), and cells wereunbound using a pair of forceps and centrifuged (1,200 rpm, 5 minutes),the supernatant was removed, followed by treatment with 3 ml of aTris-ammonium chloride buffet (pH 7.65) for 1 to 2 minutes to eliminateerythrocytes. The remaining cells were further washed three times withMEM and used for cell fusion.

(4) Preparation of Mouse Myeloma Cell

An 8-azaguanine-resistant mouse myeloma cell line, P3X63Ag8U.1 (ATCCCRL-1597, hereinafter referred to as “P3-U1”), was cultured and used asthe parent line in cell fusion.

(5) Preparation of Hybridoma Cell

The spleen cells and myeloma cells obtained in (3) and (4) in ReferenceExample 1 were mixed to a ratio of 10:1, followed by centrifuging (1,200rpm, 5 minutes) to remove the supernatant, 0.5 ml of a polyethyleneglycol solution (a solution containing 2 g of polyethylene glycol-1000,2 ml of MEM and 0.7 ml of DMSO) was added to the thus precipitated cellsper 10⁸ of spleen cells at 37° C., followed by thoroughly suspending.Thereafter, 1 to 2 ml of MEM was added several times at 1 to 2 minuteintervals, and the final volume was adjusted to 50 ml with MEM. Afterremoving the supernatant by centrifugation (900 rpm, 5 minutes), theprecipitate was suspended in 100 ml of HAT medium, dispensed in 100μl/well into a 96 well microtiter plate (manufactured by SumitomoBakelite), followed by culturing in a 5% CO₂ incubator at 37° C. for 10to 14 days. Using wells in which propagation of the fused cell wasobserved, binding activity to the hCCR4 partial peptide (Compound 2) inthe culture supernatant was measured by ELISA (Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, Chapter 14 (1988), MonoclonalAntibodies: Principles and Practice, Academic Press Limited (1966),etc.). Each well in which the activity was confirmed was cloned by atotal of 2 times of limiting dilution, once by changing the medium tothe HT medium and then changing the medium to the normal medium. In thisway, a hybridoma cell KM2160 which produces the mouse antibody KM2160was obtained. KM2160 specifically reacted with the hCCR4 partial peptide(Compound 2).

Reference Example 2 Preparation of Anti-CCR4 Chimeric Antibody

1. Isolation and Analysis of cDNA Encoding V Region of Anti-CCR4 MouseAntibody:

(1) Preparation of mRNA from Hybridoma Cell which Produces Anti-CCR4Mouse Antibody

A mRNA was prepared from the hybridoma cell KM2160 described inReference Example 1. About 48 μg of mRNA was prepared from 8×10⁷ cellsof the hybridoma cell KM2160 using a mRNA preparation kit, Fast TrackmRNA Isolation Kit (manufactured by Invitrogen) according to themanufacture's instructions.

(2) Preparation of H Chain and L Chain cDNA Library of Anti-CCR4 MouseAntibody

cDNA having EcoRI-NotI adapters on both termini was synthesized from 5μg of the KM2160 mRNA obtained in 1(1) of Reference Example 2 using cDNASynthesis kit (manufactured by Amersham Pharmacia Biotech) according tothe manufacture's instructions. The thus prepared cDNA was dissolved in20 μl of sterile water and fractionated by agarose gel electrophoresis,and about 1.5 kb cDNA fragments corresponding to the H chain of IgG typeantibody and about 1.0 kb cDNA fragments corresponding to the L chain ofκ type were respectively recovered using QIAquick Gel Extraction Kit(manufactured by QIAGEN). Next, using λZAPII PredigestedEcoRI/CIAP-Treated Vector Kit (manufactured by Stratagene), each of 0.1μg of the about 1.5 kb cDNA fragments and 0.1 μg of the about 1.0 kbcDNA fragments was linked to 1 μg of the λZAPII vector which had beendigested with a restriction enzyme EcoRI and terminus-dephosphorylatedwith Calf Intestine Alkaline Phosphatase according to the manufacture'sinstructions. Into λ phage, 2.5 μl of each reaction solution after theligation was packaged using GigapackIII Gold Packaging Extract(manufactured by Stratagene) according to the manufacture'sinstructions, and then Escherichia coli XL1-Blue (Biotechniques, 5, 376(1987)) was infected with an appropriate amount of the phage to obtain9.3×10⁴ of phage clones as the H chain cDNA library of KM2160 and7.4×10⁴ of phage clones as the L chain cDNA library. Thereafter, eachphage was fixed on a nylon membrane filter Hybond-N+ (manufactured byAmersham Pharmacia Biotech) according to the manufacture's instructions.

(3) Cloning of H Chain and L Chain cDNAs of Anti-CCR4 Mouse Antibody

Using ECL Direct Nucleic Acid Labeling and Detection System(manufactured by Amersham Pharmacia Biotech), according to themanufactures instructions, clones on the nylon membrane filters of theKM2160 H chain cDNA library and L chain cDNA library prepared in 1(2) ofReference Example 2 were detected using cDNA of the C region of a mouseantibody (H chain is a BamHI-EcoRI fragment of mouse Cγ1 cDNA (EMBO J.,3, 2047 (1984)), L chain is a HpaI-EcoRI fragment of Cκ cDNA (Cell, 22,197 (1980)) as the probe, and phage clones strongly bound to the probewere obtained as 10 clones for each of the H chain and the L chain.Next, each phage clone was converted into plasmid by the in vivoexcision method Using λZAPII Cloning Kit according to the manufacture'sinstructions (manufactured by Stratagene). Using BigDye Terminator CycleSequencing FS Ready Reaction Kit (manufactured by PE Biosystems), thenucleotide sequence of cDNA contained in each plasmid obtained in thismanner was analyzed by a DNA sequencer ABI PRISM 377 of the samemanufacturer according to the manufacture's instructions. As a result, aplasmid pKM2160H4 containing a full length functional H chain cDNA and aplasmid pKM2160L6 containing a full length L chain cDNA, in which an ATGsequence considered to be the initiation codon is present in the5′-terminal of cDNA, were obtained.

(4) Analysis of Amino Acid Sequence of V Region of Anti-CCR4 MouseAntibody

A full nucleotide sequence of the H chain V region contained in theplasmid pKM2160H4, a full amino acid sequence of the H chain V regiondeduced therefrom, a full nucleotide sequence of the L chain V regioncontained in the plasmid pKM2160L6 and a full amino acid sequence of theL chain V region deduced therefrom are represented by SEQ ID NOs:61, 62,63 and 64, respectively. Based on the comparison with sequence data ofknown mouse antibodies (Sequences of Proteins of Immunological Interest,US Dept. Health and Human Services (1991)) and the comparison with theresults of analysis of the H chain and L chain N-terminal amino acidsequences of the purified anti-CCR4 mouse antibody KM2160 carried outusing a protein sequencer (PPSQ-10, manufactured by Shimadzu), it wasfound that each cDNA thus isolated is a full length cDNA which encodesthe anti-CCR4 mouse antibody KM2160 containing secretory signalsequences which are amino acids of positions 1-19 in the amino acidsequence represented by SEQ ID NO:15 in the H chain and amino acids ofpositions 1-19 in the amino acid sequence represented by SEQ ID NO:25 inthe L chain.

Next, novelty of the amino acid sequences of the V regions of H chainand L chain of the anti-CCR4 mouse antibody KM2160 was examined. UsingGCG Package (version 9.1, manufactured by Genetics Computer Group) asthe sequence analyzing system, amino acid sequence data base of knownproteins were searched by BLAST method (Nucleic Acids Res., 25, 3389(1997)). As a result, completely coincided sequences were not found forboth of the H chain and L chain, so that it was confirmed that the Hchain V region and L chain V region of the anti-CCR4 mouse antibodyKM2160 are novel amino acid sequences.

Also, CDRs of the H chain V region and L chain V region of the anti-CCR4mouse antibody KM2160 were identified by comparing with amino acidsequences of known antibodies. Amino acid sequences of CDR1, CDR2 andCDR3 in the H chain V region of the anti-CCR4 mouse antibody KM2160 arerepresented by SEQ ID NOs:1, 2 and 3, respectively, and amino acidsequences of CDR1, CDR2 and CDR3 in the L chain V region in SEQ IDNOs:5, 6 and 7, respectively.

2. Stable Expression of Anti-CCR4 Chimeric Antibody Using Animal Cell

(1) Construction of Anti-CCR4 Chimeric Antibody Expression VectorpKANTEX2160

An anti-CCR4 chimeric antibody expression vector pKANTEX2160 wasconstructed as follows, using a humanized antibody expression vectorpKANTEX93 which expresses a human IgG1 and κ type antibody and theplasmids pKM2160H4 and pKM2160L6 obtained in 1(3) of Reference Example2.

A synthetic DNA having the nucleotide sequences represented by SEQ IDNOs:65 and 66 was designed in order to obtain the H chain V region cDNAof KM2160 by PCR, and another synthetic DNA having the nucleotidesequences represented by SEQ ID NOs:67 and 68 for obtaining the L chainV region cDNA. Each synthetic DNA contains a restriction enzymerecognizing sequence in its 5′-terminal for its cloning into pKANTEX93,and synthesis of the DNA was entrusted to Genset Inc. The plasmidpKM2160H4 (20 ng) obtained in 1(3) of Reference Example 2 was added to abuffer containing 50 μl of PCR Buffer #1 attached to KOD DNA Polymerase(manufactured by TOYOBO), 0.2 mM dNTPs, 1 mM magnesium chloride and 0.5μM of the synthetic DNA having the nucleotide sequences represented bySEQ ID NOs:11 and 12, and the mixture was heated at 94° C. for 3minutes. After 2.5 units of KOD DNA Polymerase (manufactured by TOYOBO)were added, the mixture was subjected to 25 cycles of the reaction eachcycle consisting of heating at 94° C. for 30 seconds, at 58° C. for 30seconds and at 74° C. for 1 minute, using a DNA thermal cycler GeneAmpPCR System 9600 (manufactured by PERKIN ELMER). In the same manner, 20ng of the plasmid pKM2160L6 obtained in 1(3) of Reference Example 2 wasadded to a buffer containing 50 μl of PCR Buffer #1 attached to KOD DNAPolymerase (manufactured by TOYOBO), 0.2 mM dNTPs, 1 mM magnesiumchloride and 0.5 μM of the synthetic DNA having the nucleotide sequencesrepresented by SEQ ID NOs:67 and 68, and PCR was carried out in the samemanner as described above. The reaction solution (10 μl) was subjectedto agarose gel electrophoresis, and then an H chain V region PCR productof about 0.46 kb and an L chain V region PCR product of about 0.43 kbwere each recovered using QIAquick Gel Extraction Kit (manufactured byQIAGEN).

Next, 0.1 μg of DNA obtained by digesting a plasmid pBluescript SK(−)(manufactured by Stratagene) with a restriction enzyme SmaI(manufactured by Takara Shuzo) and about 0.1 μg of each of the PCRproducts obtained above were added to sterile water to give a finalvolume of 7.5 μl, and 7.5 μl of the solution I of TAKARA DNA LigationKit Ver. 2 (manufactured by Takara Shuzo) and 0.3 μl of a restrictionenzyme SmaI were added thereto, and the mixture was allowed to react at22° C. overnight. Using the resulting recombinant plasmid DNA solution,E. coli DH5α (manufactured by TOYOBO) was transformed. Each plasmid DNAwas prepared from the transformant clones and subjected to the reactionusing BigDye Terminator Cycle Sequencing FS Ready Reaction Kit(manufactured by PE Biosystems) according to the manufacture'sinstructions, and the nucleotide sequence was analyzed by a DNAsequencer ABI PRISM 377 of the same manufacturer. Thus, the plasmidspKM2160VH41 and pKM2160VL61 shown in FIG. 15 having the desirednucleotide sequences were obtained.

Next, 3 μg of the humanized antibody expression vector pKANTEX93 and 3μg of the pKM2160VH41 obtained above were added to a buffer containing30 μl of 10 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride and 1 mM DTT,10 units of a restriction enzyme ApaI (manufactured by Takara Shuzo)were added thereto, and the mixture was allowed to react at 37° C. for 1hour. The reaction solution was subjected to ethanol precipitation, andthe resulting precipitate was added to a buffer containing 10 μl of 50mM Tris-HCl (pH 7.5), 100 mM sodium chloride, 10 mM magnesium chloride,1 mM DTT, 100 μg/ml BSA and 0.01% Triton X-100, 10 units of arestriction enzyme NotI (manufactured by Takara Shuzo) were addedthereto, and the mixture was allowed to react at 37° C. for 1 hour. Thereaction mixture was fractionated by agarose gel electrophoresis, andabout 12.75 kb and about 0.44 kb ApaI-NotI fragments of pKANTEX93 andpKM2160VH41, respectively, were recovered. The thus obtained twofragments were linked using TAKARA DNA Ligation Kit Ver. 2 according tothe manufacture's instructions, and E. coli DH5α (manufactured byTOYOBO) was transformed using the resulting recombinant plasmid DNAsolution. Each plasmid DNA was prepared from the transformant clones andconfirmed by a restriction enzyme treatment to thereby obtain a plasmidpKANTEX2160H shown in FIG. 16, in which about 0.44 kb of the desiredApaI-NotI fragment had been inserted.

Next, 3 μg of the pKANTEX2160H and 3 μg of the pKM2160VL61 obtainedabove were added to a buffer containing 50 mM Tris-HCl (pH 7.5), 100 mMsodium chloride, 10 mM magnesium chloride, 1 mM DTT and 100 μg/ml ofBSA, the solution was adjusted to give a total volume of 30 μl, 10 unitsof a restriction enzyme BsiWI (manufactured by New England Biolabs) wasadded thereto, and the mixture was allowed to react at 55° C. for 1hour. Then, a restriction enzyme EcoRI (manufactured by Takara Shuzo)was added thereto, and the mixture was allowed to react at 37° C. for 1hour. The reaction mixture was fractionated by agarose gelelectrophoresis, and about 13.20 kb and about 0.41 kb EcoRI-BsiWIfragments of pKANTEX2160H and pKM2160VL61, respectively, were recovered.The thus obtained two fragments were linked using TAKARA DNA LigationKit Ver. 2 according to the manufacture's instructions, and E. coli DH5α(manufactured by TOYOBO) was transformed using the resulting recombinantplasmid DNA solution. Each plasmid DNA was prepared from thetransformant clones and confirmed by a restriction enzyme treatment tothereby obtain a plasmid pKANTEX2160 shown in FIG. 17, in which about0.41 kb of the desired EcoRI-BsiWI fragment had been inserted. When theplasmid was subjected to the reaction using BigDye Terminator CycleSequencing FS Ready Reaction Kit (manufactured by PE Biosystems)according to the manufacture's instructions, and the nucleotide sequencewas analyzed by a DNA sequencer ABI PRISM 377 of the same manufacturer,it was confirmed that the desired plasmid into which cDNA encoding theKM2160 H chain and L chain V regions had been cloned was obtained.

(2) Stable Expression of Anti-CCR4 Chimeric Antibody Using Animal Cell

The anti-CCR4 chimeric antibody was expressed in animal cells asdescribed below using the anti-CCR4 chimeric antibody expression vectorpKANTEX2160 obtained in 2(1) of Reference Example 2.

The plasmid pKANTEX2160 was converted into a linear form by digestingwith a restriction enzyme AatII (manufactured by TOYOBO) and 10 μgthereof was introduced into 4×10⁶ cells of rat myeloma cell line YB2/0(ATCC CRL1662) by electroporation (Cytotechnology, 3, 133 (1990)), andthe cells were suspended in 40 ml of H-SFM (manufactured by GIBCO-BRL)medium (supplemented with 5% FCS) and dispensed in 200 μl/well into a 96well microtiter plate (manufactured by Sumitomo Bakelite). Twenty-fourhours after incubation at 37° C. in a 5% CO₂ incubator, G418 was addedto give a concentration of 1 mg/ml, followed by culturing for 1 to 2weeks. A culture supernatant was recovered from a well in which a colonyof G418-resistant transformant appeared and became confluent, andantigen-binding activity of the anti-CCR4 chimeric antibody in thesupernatant was measured by ELISA shown in 2(3) of Reference Example 2(a peroxidase-labeled goat anti-human IgG(γ) antibody was used as thesecondary antibody).

In order to increase the expressed amount of the antibody using a dhfrgene amplification system, the transformant in a well where expressionof the anti-CCR4 chimeric antibody was found in the culture supernatantwas suspended to give a density of 1 to 2×10⁵ cells/ml in H-SFM mediumcontaining 1 mg/ml G418 and 50 nM methotrexate (hereinafter referred toas “MTX”: manufactured by Sigma) which is the inhibitor of a dhfr geneproduct dihydrofolate reductase, and the suspension was dispensed in 1ml into wells of a 24 well plate (manufactured by Greiner). The mixturewas cultured at 37° C. for 1 to 2 weeks in a 5% CO₂ incubator, so that atransformant showing resistance to 50 nM MIX was induced. When thetransformant became confluent in a well, antigen-binding activity of theanti-CCR4 chimeric antibody in the culture supernatant was measured byELISA shown in 2(3) of Reference Example 2. Regarding the transformantsin wells where expression of the anti-CCR4 chimeric antibody was foundin culture supernatants, the MTX concentration was increased to 100 nMand then to 200 nM in the same manner to finally obtain a transformantwhich can grow in H-SFM medium containing 1 mg/ml of G418 and 200 nM ofMTX and can also highly express the anti-CCR4 chimeric antibody. Thethus obtained transformant was subjected to single cell isolation(cloning) by two times of limited dilution assay, and a transformantclone having the highest anti-CCR4 chimeric antibody expression wasnamed KM2760. The expressed amount of the anti-CCR4 chimeric antibody byKM2760 was about 5 μg/10⁶ cells/24 hours. In addition, the antibody Hchain C region of KM2760 belongs to human IgG1 subclass. KM2760 has beeninternationally deposited as FERM BP-7054 on Feb. 24, 2000, in NationalInstitute of Bioscience and Human Technology, Agency of IndustrialScience and Technology, the Ministry of International Trade and Industry(present name: International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology) (Higashi 1-1-3,Tsukuba-shi, Ibaraki Prefecture, Japan (present address: AIST TsukubaCentral 6, 1-1, Higashi 1-Chome Tsukuba-shi, Ibaraki-ken 305-8566Japan)).

Reference Example 3 Establishment of hCCR4-High-Expressing Cell

(1) Construction of Expression Vector CAG-pcDNA3 for Animal Cell

An expression vector was constructed as descried below by producing anexpression vector (CAG-pcDNA3) in which the promoter region of anexpression vector for animal cell, pcDNA3 (manufactured by INVITROGEN),was changed from cytomegalovirus (CMV) promoter to CAG (AG (modifiedchicken β actin) promoter with CMV-IE enhancer), and inserting the CCR4gene into the vector.

pcDNA3 (5 μg) was allowed to react with a restriction enzyme NruI(manufactured by Takara Shuzo) at 37° C. for 1 hour, and then DNAfragments were recovered by ethanol precipitation. Next, they wereallowed to react with a restriction enzyme HindIII (manufactured byTakara Shuzo) at 37° C. for 1 hour and then fractionated by agarose gelelectrophoresis to recover a DNA fragment of about 5.8 kb containing noCMV promoter region. Plasmid CAG-pBluescript IIKS(+) (3 μg) having CAGpromoter (Nuc. Acid. Res., 23, 3816 (1995)) region was allowed to reactwith a restriction enzyme SalI (manufactured by Takara Shuzo) at 37° C.for 1 hour and then DNA fragments were recovered by ethanolprecipitation. They were blunt-ended with DNA Blunting Kit (manufacturedby Takara Shuzo), further allowed to react with HindIII at 37° C. for 1hour, and then fractionated by agarose gel electrophoresis to recover aDNA fragment of about 1.8 kb containing the CAG promoter region. Thethus recovered respective DNA fragments were ligated using DNA LigationKit (manufactured by Takara Shuzo), and E. coli DH5α was transformedusing the resulting recombinant plasmid DNA to obtain plasmidCAG-pcDNA3.

(2) Construction of hCCR4 Expression Vector

An hCCR4 expression vector was constructed as described below by usingthe CAG-pcDNA3 obtained in 3(1) of Reference Example 2 and hCCR4DNA-inserted pcDNA3 (CCR4/pcDNA3). Both of the CAG-pcDNA3 andCCR4/pcDNA3 were allowed to react with HindIII at 37° C. for 1 hour andDNA fragments were recovered by ethanol precipitation. Next, they wereallowed to react with BglII (manufactured by Takara Shuzo) at 37° C. for1 hour and then fractionated by agarose gel electrophoresis to recover aDNA fragment of about 2.0 kb containing the CAG promoter region and aDNA fragment of about 5.5 kb containing the hCCR4 gene region.Thereafter, plasmid CAG-CCR4/pcDNA3 was obtained using both of the DNAfragments in the same manner as in 3(1) of Reference Example 2.

(3) Expression of hCCR4 in Animal Cell

The plasmid was introduced into animal cells by electroporation in thesame manner as described in 2(2) of Reference Example 2. EL-4 cells(ATCC TIB-39) were suspended in PBS(−) (manufactured by GIBCO-BRL) togive a density of 1×10⁷ cells/500 μl, 10 μg of the CAG-CCR4/pcDNA3obtained in 3(2) of Reference Example 2 was added thereto, and themixture was incubated in ice for 10 minutes and then put into a cuvettefor exclusive use (manufactured by Bio-Rad) to carry out geneintroduction at 260 V and 500 μFD. After the mixture was furtherincubated in ice for 10 minutes, the cells were suspended in 200 ml of10% FCS-RPMI medium and dispensed at 200 μl/well into a 96 well platefor cell culturing. Twenty-four hours after culturing, 100 μl of theculture supernatant was removed from each well, and 10% FCS-RPMI mediumcontaining 1 mg/ml of G418 was dispensed at 100 μl/well to give a finalconcentration of 0.5 mg/ml. Two weeks thereafter, single clones ofbetween 10 and 100 were selected and cultured again.

(4) Selection of hCCR4-High-Expressing Cell

They were selected by an immunofluorescent method using KM2160 preparedin (5) of Reference Example 1. Into a 96 well U shape plate, 2×10⁵ cellsof each of selected several tens of the gene-introduced clones wasdispensed. KM2160 labeled with biotin by a known method (Enzyme AntibodyMethod, published by Gakusai Kikaku) was diluted to 5 μg/ml with abuffer for FACS (1% BSA-PBS, 0.02% EDTA, 0.05% NaN₃, pH 7.4), human IgG(manufactured by Welfide) was diluted to 3.75 mg/ml to preventnonspecific staining, each of the thus diluted antibody solution wasdispensed at 200 μl/well, and the mixture was allowed to react in icefor 30 minutes. As a negative control, biotinylated anti-IL-5R antibody(WO 97/10354) was used at the same concentration. After washing twicewith 200 μl/well of the buffer, streptoavidin-PE (manufactured by BectonDickinson Japan) was dispensed at 20 μl/well. Thirty minutes after thereaction in ice in the dark, the cells were washed three times with 200μl/well and finally suspended to 500 μl, and the fluorescence intensitywas measured by a flow cytometer to select one cell line having thehighest fluorescence intensity. The cell line having the highestfluorescence intensity was used as CCR4/EL-4.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one of skill in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof. All references cited hereinare incorporated in their entirety.

This application is based on Japanese application No. 2001-265144 filedon Aug. 31, 2001, the entire content of which is incorporated hereintoby reference.

What is claimed is:
 1. A method for treating cancer in a patient, said method comprising administering to a patient in need thereof an effective amount of a human CDR-grafted antibody or antibody fragment thereof which reacts with an extracellular region of human CC chemokine receptor 4 (CCR4), wherein the cancer cells of said cancer express CCR4, and wherein the antibody or antibody fragment comprises an antibody heavy chain (H chain) variable region (V region) comprising SEQ ID NO: 4, 9, 10, 11, 38, 39, 40 or 41; and an antibody light chain (L chain) V region comprising SEQ ID NO:
 14. 2. The method according to claim 1, wherein the human CDR-grafted antibody or antibody fragment thereof comprises an antibody heavy chain (H chain) variable region (V region) comprising SEQ ID NO: 9 or 10; and an antibody light chain (L chain) V region comprising SEQ ID NO:
 14. 3. The method according to claim 1, wherein the cancer is a blood cancer.
 4. The method according to claim 3, wherein the blood cancer is a leukemia or a lymphoma. 